Mice
Nelfbfl/fl mice61 were a gift from R. Li. AID-cre mice62 were provided by M. Muschen and were used as heterozygotes. Experimental mice were generated by intercrossing AID-cre and Nelfbfl/fl mice to generate Nelfbfl/fl;AID-cre and control Nelfbfl/+;AID-cre mice. Both male and female mice were used in experiments. Splenic GCB cells were sorted as the B220+PNAhiCD95hi population 7 days after intraperitoneal injection of sheep red blood cells followed by mutation sequencing analysis. Animal studies were performed without randomization, investigator blinding or previous analyses of sample size. All animal studies were conducted in accordance with national guidelines and were approved by the Yale Institutional Animal Care and Use Committee.
Cell culture
Ramos cells and cell lines derived from Ramos cells were grown in RPMI-1640 (Gibco, 11875) supplemented with 10% FBS (Gemini) and 0.5 mg ml–1 penicillin–streptomycin–glutamine (Gibco, 10378016) at 37 °C, 5% CO2. Ramos cells have been used extensively for the study of SHM factors and mechanisms60,63,64,65,66,67,68,69,70,71,72,73,74,75,76. 293T cells were grown at 37 °C, 5% CO2 in DMEM (Gibco, 10566) supplemented in the same manner as Ramos cells. CH12F3 cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Gibco), 1× MEM non-essential amino acids, 10 mM HEPES, 2 mM Glutamax, 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol and penicillin and streptomycin77.
Antibodies and western blots
Polyclonal anti-INTS8 antibody (AB2474) was generated by immunizing rabbits with a peptide spanning amino acids 26–44 of mouse INTS8 (EFLLEESLLEKHLRKACPD), a region that is highly conserved in human INTS8, followed by affinity purification against the peptide. It was provided for this study by R. Shiekhattar. Commercially available antibodies used in this study are listed in the Reporting summary. Raw western blot gel files are provided in Supplementary Fig. 1.
Generation of human GCB cell lines
Ex vivo human tonsillar GCB cells (hGCB-Bcl2-Bcl6-Cas9) and YK6-CD40Lg-IL21 feeder cells (hereafter referred to as YK6 cells) were obtained from M. Müschen and were based on a previously described system78,79. Cell culture, GaLV-pseudotyped lentiviral production and transduction of GCB cells with GaLV-pseudotyped lentivirus were performed as previously described79. YK6 cells were maintained in RPMI medium supplemented with 10% FBS in 150 × 25 mm tissue-culture-treated dishes. GCB cells were cultured in Advanced RPMI medium supplemented with 20% FBS on irradiated YK6 feeder cells. For feeder preparation, approximately 4–8 × 107 YK6 cells were irradiated with 30 Gy and cryopreserved at approximately 4 × 106 cells per vial, corresponding to the number of feeder cells required for one 12-well plate. One day before GCB cell culture, a vial of irradiated YK6 cells was thawed, plated in a 12-well plate and allowed to adhere overnight. HEK293T cells were used for GaLV-pseudotyped lentiviral production. Approximately 5 × 106 cells were seeded in a 10 cm dish 1 day before transfection. On the day of transfection, 33 μl TransIT-293 reagent was mixed with 1 ml Opti-MEM and incubated at room temperature (RT) for 5–10 min. Subsequently, 2.8 μg GaLV MTR, 8.3 μg pCMVDeltaR8.91 and 11 μg of the lentiviral construct (either superDIVAC-GFP7, a SHM reporter containing HTS7–GFP flanked by the strong superDIVAC SHM-targeting element27 or Tet3G-AID7.3, which expresses doxycycline-inducible AID7.3) were added to the mixture, vortexed briefly and incubated at RT for an additional 15–30 min. The transfection mixture was then added dropwise to HEK293T cells. Viral supernatants were collected 36–48 h after transfection and stored at −80 °C until use. For lentiviral transduction, HEPES and Polybrene were added to fresh or thawed viral supernatant to final concentrations of 25 mM and 10 μg ml–1, respectively. GCB cell cultures were centrifuged at 400g for 5 min at RT and the supernatant was carefully removed, leaving approximately 100 μl of medium in the well containing GCB and feeder cells. Subsequently, 500 μl viral supernatant was added to the well of a 12-well plate, followed by spin infection at 1,500g for 90 min at 32 °C. Cells were returned to the incubator for 4–12 h and the viral supernatant was replaced with fresh Advanced RPMI medium containing 20% FBS after centrifuging the plate at 400g for 5 min at RT. Two to three days after transduction, GFP-positive cells were isolated by FACS following superDIVAC-GFP7 transduction. Cells transduced with Tet3G-AID7.3 were selected using puromycin.
Generation of degron cell lines
Degron cell lines were generated using RASH-1C or CH12F3 cells. For construction of NELFA degron, NELFC/D degron, MLLT1 degron and INTS6 degron cells, plasmids expressing the sgRNA targeting the stop codon region and donor plasmids containing left and right homology arms attached to HA-FKBP12(F36V)-mScarlet sequences80 were delivered into RASH-1C or human GCB cells previously transduced with superDIVAC-GFP7 and Tet3G-AID7.3. For construction of SPT5 degron, INTS1 degron, INTS5 degron, INTS8 degron, INTS10 degron and INTS11 degron cells, plasmids expressing the sgRNA targeting the start codon region and donor plasmids containing left and right homology arms attached to HA-FKBP12(F36V)-mScarlet sequences were delivered into RASH-1C cells. For NELFA degron and NELFC/D degron CH12F3 cells, plasmids expressing sgRNA targeting mouse NelfA and NelfCD stop codon regions and donor plasmids containing left and right homology arms attached to HA-FKBP12(F36V)-mScarlet sequences80 were delivered into CH12F3 cells. Homemade 1 M buffer was used for electroporation according to a previously described method81. Electroporation was done by using Amaxa Nucleofector II (Lonza) with program O-006 according to the manufacturer’s instructions. Five days later, mScarlet-positive cells were sorted into a 96-well plate to obtain single-cell clones. Expanded clones were cultured for 15 days in a 96-well plate and then analysed by FACS to detect mScarlet signals and then validated by Sanger sequencing. The sequences of oligonucleotides used in this study are provided in Supplementary Table 1.
Identification of rearranged V(D)J sequences in NELFA degron human GCB cells
RNA sequencing was performed in three independent NELFA degron human GCB clones. Counts on IGHV3-23 and IGKV3-20 were observed in all three clones. Primers targeting upstream of the variable (V) region and downstream of the joining (J) segments were designed, and Sanger sequencing was performed to confirm the sequences of the rearranged V(D)J regions. The sequences of IGHV and IGKV analysed by mutation sequencing are provided in Supplementary Table 2.
Generation of MLLT1d/3KO and INTS6d/6LKO cell lines
Guide RNA targeting human MLLT3 was cloned into a vector expressing both sgRNA and blue fluorescence protein (BFP) and then delivered into MLLT1 degron cells by electroporation. Guide RNA targeting human INTS6L was delivered into INTS6 degron cells. One day after electroporation, GFP, mCherry and BFP triple-positive cells were sorted into 96-well plates for single-KO clone selection. Successful KO clones were validated by Sanger sequencing.
Degron cell line cell culture analysis
In a typical experiment, degron RASH-1C cells were treated with the degradation inducer dTAGV-1 or its negative control compound (NEG) for 2–4 h to eliminate the protein of interest, followed by doxycycline induction of AID to initiate SHM for 1–5 days. dTAGV-1 or NEG were maintained in the cultures throughout the doxycycline induction period. SHM was measured by loss of GFP fluorescence or surface IgM expression or by high-throughput mutation sequencing of IGHV, IGLV or HTS7 upstream of the GFP reporter.
Reconstitution in degron or KO cells
NELFA or MLLT1 or their mutants were cloned into a retroviral plasmid pMSCV. INTS8 or its mutant was cloned into a lentiviral plasmid PLV-EF1α-puro. 293T cells in 6-well plates were grown to 50–80% confluence and then transduced with 1 µg retroviral plasmid and 1 µg packaging plasmid pkat2 or 1 μg lentiviral plasmid, 0.6 μg psPAX2 (Addgene, 12260) and 0.4 μg pMD2.G (Addgene, 12259) with JetPrime reagent (Polyplus, 114-07) according to the manufacturer’s protocol. At 48 h after transfection, retroviral or lentiviral-containing media were collected and filtered through a 0.45 μm filter before being used to infect cells. Around 1 million Ramos or RASH-1C degron cells were mixed with 100 μl filtered virus and incubated at 37 °C, 5% CO2, for 30 mins before being transferred into a 6-well plate. Seven days after puromycin selection, cells stably expressing WT or mutant proteins were treated with either NEG or dTAGV-1 followed by doxycycline treatment.
IgM and GFP loss assay
For the GFP-loss and IgM-loss assays, RASH-1C-derived cells were treated with or without 200 ng ml–1 doxycycline for 3 days. Around 1.0 × 106 RASH-1C cells were stained with APC mouse anti-human IgM (Becton Dickenson (BD), 551062) diluted 1:100 in FACS Buffer (1× PBS with 2% FBS) and incubated for 20 min at RT. The percentage of GFP+ and GFP− cells (GFP loss), APC+ and APC− cells (IgM loss) were determined by flow cytometry. Flow cytometry data were analysed using FlowJo (v.10.6.2) software. Gating strategies are provided in Supplementary Fig. 2.
Analysis of G1-arrested cells
NELFA degron or NELFC/D degron cells were treated with 5 μM palbociclib (S1116, Selleckchem) for 1 day or 2 days. Cell cycle analyses were performed using a Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay kit (C10634, Sigma) according to the manufacturer’s manual. Cells were treated with 10 μM EdU (included in the kit) for 1 h at the end of the experiment and then collected followed by fixation, permeabilization, EdU and DAPI staining and analysis by flow cytometry.
Mutation sequencing
Analysis of mutations by DNA sequencing was performed as previously described23. In brief, gDNA was extracted using a DNeasy Blood & Tissue kit (Qiagen, 69504) according to the manufacturer’s instructions. Target regions were amplified using the oligonucleotides outlined in Supplementary Table 1. PCR reactions were performed using NEBNext High-Fidelity 2× PCR master mix (NEB) under the following conditions: 98 °C for 3 min, followed by 25 cycles at 98 °C for 10 s; 60 °C for 30 s; 72 °C for 30 s; and 72 °C for 2 min in a 25 µl reaction. PCR products were multiplexed using a NextEra XT index kit (Illumina) and sequenced using an Illumina NovaSeq X (300 cycles).
Co-immunoprecipitation
Co-immunoprecipitation was performed as previously described with modifications58. Around 40 million cells were resuspend with 1 ml nuclear extraction buffer (20 mM HEPES pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% glycerol and 1× protease inhibitor cocktail (Roche)) and rotated at 4 °C for 20 min. Nuclear pellets were then resuspended in 400 μl low-salt buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM MgCl2, 10% glycerol, 1% NP40 and 1× protease inhibitor cocktail (Roche)) containing 250 U ml–1 Benzonase nuclease (Sigma) and were sonicated 5 × 10 s at 50% amplitude at 4 °C. Samples were then incubated for 30 min on a rotator at 4 °C followed by centrifugation at maximum speed for 10 min. Supernatant was collected and mixed with 250 μl high-salt buffer (low-salt buffer containing 400 mM NaCl). Next, 2.5 μg anti-AID antibody (Invitrogen, 39-2500) or 2 μg anti-Flag antibody (Sigma, F3165) was added and samples were incubated overnight. Protein complexes were pulled down by 4 h of incubation with 25 μl Pierce Protein A/G magnetic beads (ThermoFisher, 88802) and the samples were prepared by boiling in Laemmli–SDS sample buffer for subsequent analyses by western blotting.
CSR analysis
To assess CSR from IgM to IgA in the CH12F3 cell line, cells were plated at a density of 1 × 105 per ml of medium and stimulated with anti-CD40 (1 μg ml–1, clone 1C10, BioLegend), rmIL-4 (10 ng ml–1, PeproTech) and rhTGF-b (1 ng ml–1, R&D) (CIT) for 48 h.
CUT&RUN-seq
CUT&RUN-seq was performed according to the manufacturer’s protocol using a CUTANA ChIC/CUT&RUN kit (Epicypher, 14-1048). In brief, degron cells were treated with 0.5 μM NEG (Tocris, 6915) or dTAGV-1 (Tocris, 6914) for 4 h followed by 200 ng ml–1 doxycycline treatment for 1–2 days. About 7.5 × 105 cells were collected for nucleus extraction. Isolated nuclei were bound to activated concanavalin-A-coated magnetic beads and then incubated with 0.5 μg antibodies at 4 °C overnight on a nutator. After two washes with cell permeabilization buffer, 2.5 μl pAG/MNase was added to beads resuspended in 50 μl cell permeabilization buffer and incubated for 10 min at RT. After two washes with cell permeabilization buffer, 1 μl 100 mM CaCl2 was added and then incubated at 4 °C for 2 h on a nutator. Next, 33 μl stop buffer was added and incubated at 37 °C for 10 min, and DNA was purified using SPRIselect reagent (beads). Libraries were prepared using a NEBNext Ultra II DNA Library Prep kit (NEB, E7645S) and sequenced on an Illumina NovaSeq X (paired-end, 2 ×75 bp) at an average depth of 20 million reads per sample.
TT-TimeLapse-seq
TT-TimeLapse-seq was performed as previously described33. Approximately 20 million cells per sample were labelled with 1 mM s4U (Fisher Scientific, 13957-31-8) for 5 min. Cells were collected and washed in ice-cold PBS and dissolved in Trizol. Total RNA was extracted and treated with TURBO DNase (ThermoFisher, AM2238) to deplete gDNA. Next, 2% or 5% gDNA-depleted Drosophila S2 cell spike-in RNA (5 min s4U treated) was added. RNA was then sheared by mixing with 2× RNA fragmentation buffer (150 mM Tris pH 7.4, 225 mM KCl, 9 mM MgCl2) and heated to 94 °C for 3.5 min. RNA shearing was stopped by adding EDTA (to a final concentration of 50 mM) and incubated on ice for 2 min. RNA was purified using a RNeasy Mini kit (Qiagen, 74104). Fragmented RNA was biotinylated using 10% MTSEA-biotin-XX (VWR, 89139-636) prepared in DMF. The biotinylated RNA was purified and isolated using 10 μl Dynabeads MyOne Streptavidin C1 magnetic beads (Invitrogen, 65001). s4U-biotinylated RNA was eluted by reducing the disulfide bond that formed between biotin and 4-thiouridine, thereby eluting the s4U-RNA and leaving biotin bound to the streptavidin beads using 25 μl elution buffer (100 mM DTT, 20 mM DEPC, pH 7.4, 1 mM EDTA, 100 mM NaCl and 0.05% Tween-20). TimeLapse chemistry was carried out as previously described33 to convert U to C. In brief, 20 µl eluted RNA was chemically treated with TimeLapse chemistry reagent (0.835 µl 3 M sodium acetate pH 5.2, 0.2 µl 500 mM EDTA, 1.365 µl nuclease-free water and 1.3 µl trifluoroethylamine), followed by adding freshly prepared 1.3 µl of 200 mM sodium periodate and then incubated for 1 h at 45 °C. Chemically treated RNA was purified and then incubated in a reducing buffer (10 mM DTT, 100 mM NaCl, 10 mM Tris pH 7.4 and 1 mM EDTA) at 37 °C for 30 min, followed by a second purification step. Final RNA was converted to libraries using a Takara Pico Mammalian V3 kit (Takara, 634938). Libraries were sequenced on an Illumina NovaSeq X at an average depth of 30 million reads per sample.
Protein purification and in vitro pull downs
Human AID, MLLT1 and MLLT3 (corresponding to RefSeq sequence NP_065712.1, UniProt ID Q03111 and GenBank sequence AAH36089.1, respectively) were cloned into 438 series vectors (Addgene plasmids 55219 and 55220)82 with an amino-terminal His-MBP sequence for AID or an N-terminal His tag only for MLLT1 and MLLT3, followed by a TEV protease cleavage site. The His–MBP control protein was expressed from the 438-C vector without an insert. Bacmids and virus were made as previously described83. Sf9 (His–MBP–AID) and Hi5 (MLLT1, MLLT3 and His–MBP control protein) insect cells were grown at 27 °C in ESF921 medium (Expression Systems) and collected 3–4 days after infection. Individual YEATS domains (MLLT1 amino acids 8–143, MLLT3 amino acids 1–138 and YEATS2 (UniProt Q9ULM3 201–335)) were cloned into vector 1B (Addgene, plasmid, 29653 a gift of S. Gradia) with an N-terminal His-tag followed by a TEV protease cleavage site. The T1 variant of MLLT1 YEATS was generated by site-directed mutagenesis. Proteins were expressed in Escherichia coli BL21 (RIL), which was grown to mid-log at 37 °C, shifted to 18 °C and induced with 0.5 mM IPTG overnight before collection.
All protein purification steps were performed at 4 °C or on ice. For purifying MLLT1 and MLLT3, cell pellets from 1.2 litre cultures were resuspended in 20 mM HEPES pH 7.4, 300 mM NaCl, 30 mM imidazole, 10% glycerol, 1 mM DTT, 0.284 μg ml−1 leupeptin, 1.37 μg ml−1 pepstatin A, 0.17 mg ml−1 PMSF and 0.33 mg ml−1 benzamidine (lysis buffer). Next, 1 mM MgCl2 and 5 µl salt-active nuclease (Merck, SRE0015) were added before lysis. Cells were lysed by sonication, cleared by centrifugation and filtration, and the clarified lysate was applied to a 5 ml HisTrap HP column (Cytiva) and washed sequentially with lysis buffer containing 300 mM NaCl, 1 M NaCl, 300 mM NaCl and finally 150 mM NaCl before elution with lysis buffer containing 150 mM NaCl and 500 mM imidazole. For MLLT1, protein was eluted over coupled 5 ml HiTrap SP HP and HiTrap Q HP columns (Cytiva) and eluted from the Q column with a salt gradient from 150 mM NaCl to 1 M NaCl. MLLT3 was purified similarly, except only an S column was used for ion-exchange chromatography. Fractions containing the protein of interest, as assessed by SDS–PAGE, were pooled, mixed with 1.5 mg TEV protease and 1.5 mg lambda protein phosphatase (both containing a hexahistidine tag) and dialysed into lysis buffer with 1 mM MnCl2 overnight. The next day, the dialysed sample was applied to a 5 ml HisTrap HP column (Cytiva). The flow-through fraction was concentrated in a centrifugal concentrator and applied to a HiLoad 16/600 Superose 6 pg (MLLT1) or a HiLoad 16/600 Superdex 200 pg column (MLLT3, both columns from Cytiva) equilibrated with 20 mM HEPES pH 7.4, 300 mM NaCl, 10% glycerol and 1 mM DTT. Fractions containing the protein of interest were pooled, concentrated with a centrifugal concentrator, snap-frozen as aliquots in liquid N2, and stored at −70 °C.
For purifying the His–MBP control protein (‘MBP only’), the cell pellet from 1 litre of culture was resuspended in 20 mM HEPES-KOH, pH 7.9 (at 4 °C), 500 mM NaCl, 20 mM imidazole, 1 mM TCEP, 0.284 μg ml–1 leupeptin, 1.37 μg ml–1 pepstatin A, 0.17 mg ml–1 PMSF and 0.33 mg ml–1 benzamidine (lysis buffer). Cells were lysed by sonication, cleared by centrifugation and filtration, and the clarified lysate was applied to a 5 ml HisTrap HP column (Cytiva) and washed sequentially with lysis buffer containing 300 mM NaCl, 1 M NaCl and 300 mM NaCl. Protein was eluted with lysis buffer containing a gradient of imidazole between 20 and 500 mM imidazole. Gel filtration chromatography was performed over a HiLoad 16/600 Superose 6 pg column (Cytiva) equilibrated with 20 mM HEPES-KOH, pH 7.9 (at 4 °C), 200 mM NaCl and 1 mM TCEP.
For purifying individual YEATS domains, cell pellets were resuspended in 20 mM HEPES-KOH, pH 7.6, 300 mM NaCl, 1 mM MgCl2, 20 mM imidazole, 10% glycerol, 0.5 mM TCEP, 0.284 μg ml–1 leupeptin, 1.37 μg ml–1 pepstatin A, 0.17 mg ml–1 PMSF and 0.33 mg ml–1 benzamidine (lysis buffer) supplemented with salt-active nuclease (Merck, SRE0015) and lysed by sonication. After centrifugation at 20,000g for 20 min, the supernatant was applied to a HisTrap HP column (Cytiva), washed with lysis buffer, lysis buffer containing 1 M NaCl, lysis buffer again and finally eluted with an imidazole gradient between 20 mM and 500 mM. Fractions containing the protein of interest, as assessed by SDS–PAGE, were concentrated with a centrifugal concentrator and applied to a HiLoad 16/600 Superose 6 pg gel filtration column (Cytiva) equilibrated with 20 mM HEPES-KOH, pH 7.6, 200 mM NaCl, 1 mM MgCl2, 10% glycerol and 0.5 mM TCEP. Fractions containing the protein of interest were pooled, concentrated with a centrifugal concentrator, snap-frozen as aliquots in liquid N2 and stored at −70 °C.
For purifying His–MBP–AID, cell pellets from 100 or 200 ml culture were resuspended in 20 mM HEPES-KOH, pH 7.6, 300 mM NaCl, 20 mM imidazole, 0.01% Tween 20, 5% glycerol and 0.5 mM TCEP (lysis buffer) supplemented with complete EDTA-free protease inhibitor cocktail (Roche), 1 µl salt-active nuclease (Merck SRE0015) and 1 µl benzonase (Merck 70746), disrupted by sonication and clarified by centrifugation. The clarified lysate was applied to a 1 ml HisTrap HP column (Cytiva). The column was washed sequentially with lysis buffer, lysis buffer with 800 mM NaCl, lysis buffer again, and protein was eluted with an imidazole gradient between 20 mM and 500 mM. Per pull-down reaction, 10 µg MBP–AID and 6.7 µg MBP only were coupled to 10 µl amylose resin (NEB E8021L) for 90 min rotating.
Protein-coupled amylose resin was washed 3 times with 20 mM HEPES-KOH, pH 7.5 at 4 °C, 200 mM NaCl, 2 mM MgCl2, 10% glycerol, 0.01% Tween-20 and 0.5 mM TCEP (pull-down buffer). Care was taken to ensure that the concentration of prey proteins was held between 3.5 and 4 µM throughout the pull-down assay. Prey proteins were thawed, centrifuged at 20,000g for 10 min and diluted to 4 µM in pull-down buffer and mixed with the protein-coupled amylose resin with gentle rotation for 90 min. The resin was washed four times with pull-down buffer before resuspension in SDS–PAGE loading buffer for analysis by SDS–PAGE.
Bioinformatic analyses
Mutation sequencing analysis
Sequence reads were processed using a previously described custom pipeline24. Paired-end reads were merged using the fastq-join tool, requiring at least a 10-bp overlap with a mismatch rate of ≤8%. Merged reads were aligned to their respective reference sequences using the Burrows–Wheeler aligner with default settings. Python utility Pysamstats was used to compute statistics against reference sequence positions based on the aligned BAM files. Position-wise statistics were generated for reads with a mapping quality of at least 60 and a minimum base quality of 30. For each sample, the aligned BAM file was converted to TSV format using the Java-based utility sam2tsv. An in-house AWK script generated per-read summary files for each observed variation. Only nucleotide bases with a Phred quality score of ≥30 were considered for point mutations. We then identified WRC sites (AID hotspots) from the position-based statistics files generated by Pysamstats using R script.
CUT&RUN-seq analysis
Paired-end reads were aligned to a modified version of the human genome (hg38) using Bowtie2 with local, very-sensitive alignment settings to ensure high-quality mapping. All downstream analyses were performed using the properly mapped read pairs. We merged two or three replicates and used deepTools (v.3.5.1) to generate RPKM-normalized, input-subtracted (IgG control) bigwig files. These bigwig files were subsequently used to create all coverage profiles and other metaplots. For AID CUT&RUN-seq, raw counts were obtained for all genes from AID CUT&RUN-seq for both doxycycline and no-doxycycline treated samples. AID CUT&RUN-seq experiments for ELOF1-degron, NELFA-degron, MLLT1-degron and MLLT1d/3KO cells were performed in parallel and share the same no-dox control data (Figs. 2g,h and 4a–d, and Extended Data Figs. 3g and 8a,b)24. Differential analysis was performed using DESeq2 to identify statistically peaks, defined as those with a log2 fold change of >2 and P ≤ 0.01. All CUT&RUN data presented in this study had two or three biological replicates for each condition.
TT-TimeLapse-seq analysis
TT-TimeLapse-seq reads from degron cells treated with NEG or dTAGV-1 (two or three biological replicates each), containing spiked-in RNA from Drosophila cells, were processed using the Snakemake implementation84 of the TT-TimeLapse pipeline as previously described33. In brief, the reads were trimmed to remove adaptor sequences using Cutadapt and were mapped to Bowtie2. Bam files containing uniquely mapped reads were sorted and indexed using Samtools. We used deepTools (v.3.5.1) to generate metaplots from the RPKM-normalized and spike-in scaled bigwig files. The spike-in scaling factors were calculated using DESeq2.
To assess differential RNA synthesis between the NEG-treated and dTAGV-1-treated samples, the cutadapt trimmed reads were mapped to a combined hg38 and dm6 genome using HISAT2 with default parameters and U-to-C mutation calls. Normalized mutation-specific coverage tracks were generated and visualized using IGV85.
Quantification and statistical analysis
Data were subjected to statistical analysis and plotted using GraphPad Prism. Single comparisons were performed using two-tailed Student’s t-tests, whereas multiple comparisons were assessed by two-way ANOVA. Specific numbers (n) of biological replicates are provided in the figure legends.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
