GSM5292746: RNAseq.AC7.RNA.SMARCE1_KO.2000nM.dBRD9A.Rep1b; Homo sapiens; RNA-Seq
ChIP-seq protocol: Cells were trypsinized, washed with room temperature PBS twice to remove trypsin, and divided into 40 million-cell aliquots. Cells were fixed using 1% formaldehyde (Sigma Aldrich) for 10 min at 37 °C with constant stirring to keep cells in solution, and finally quenched with 125 mM glycine for 5 min at 37 °C. Quenched cells were washed with cold PBS, and stored in 10-million aliquots at −80 °C until they were used. 10 millions cells were used per epitope in subsequent chromatin immunoprecipitations. Following nuclei extraction, chromatin was sonicated using a Covaris E220 Focused-Ultrasonicator system. Sonicated chromatin was cleared by centrifugation for 10 min at 10,000 r.p.m. (15,000 g). Soluble fractions were incubated with the indicated antibodies (see antibody list) overnight at 4°C, and captured using Protein G Dynabeads (Thermo Fisher). All ChIP-seq samples received 20 ng of spike-in Drosophila chromatin (Active Motif), and 2 µg spike-in antibody (Active Motif) for normalization post sequencing. Antibody-chromatin complexes were captured with Protein G Dynabeads (Thermo Fisher), washed extensively, eluted, and treated with RNase A (Roche) for 30 min at 37 °C and Proteinase K (Life Technologies) for 3 h at 65 °C. ChIP DNA samples were extracted using SPRI beads (Beckman Coulter Agencourt AMP Xpure), washed with 80% Ethanol, and eluted using 0.1X TE. ChIP DNA samples were stored at -20C before library prep were initiated. All ChIP seq libraries were processed with Illumina's NEBNext Ultra II DNA library Prep Kit (Followed standard protocols). All ChIP seq libraries were sequenced on the Illumina Next-seq 500 with 75 bp read single-end sequencing. ChIP-sequencing libraries were prepared with Illumina's NEBNext Ultra II DNA library Prep Kit using standard protocols. ATAC-seq protocol: ATAC-seq protocol was adapted from following protocol [cit] with slight modifications covered below. 50,000 - 100,000 cells were trypsinized, and washed with cold PBS to remove trypsin. When appropriate, lysing steps were carried out in 10X bulk. Cell pellets were resuspended in 50μl cold ATAC-seq resuspension buffer (RSB) containing 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2, supplemented with fresh NP40 (final 0.1% v/v), Tween-20 (final 0.1% v/v), Digitonin (final 0.01% v/v). Resuspended cells were incubated in lysis buffer for 3-5 min on ice. Lysis step was quenched with 1 mL of RSB supplemented with Tween-20 (final 0.1% v/v) and pelleted at 500 g for 10 min at 4°C. Cell pellets were resuspended in 50μl transposition reaction mix containing 25 μl 2X TD buffer, 2.5 μl transposase, 16.5 μl 1X PBS, 0.5 μl 1% digitonin (final 0.01% v/v), 0.5 μl 10% Tween-20 (final 0.1% v/v), and 5 μl water nuclease-free water. Transposition reaction was assembled, mixed, and incubated at 37°C for 30 min with constant shaking of 1,000 RPM on a thermomixer. To purify the tagmented DNA, Qiagen MinElute Reaction Cleanup Kit was used. Standard protocol (Buenrostro et al., 2015) [cit] with 10-12 cycles of amplification was used to amplify tagmented library. ATAC-seq libraries were sequenced on Next-seq 500 (Illumina) using 37 bp pair-end sequencing. ATAC-seq libraries were prepared using a standard protocol (Buenrostro et al., Current Protocols (2013)). RNA-seq protocol: 1,000,000 cells were trypsinized, and washed with cold PBS to remove trypsin. RNA was purified using the Qiagen RNeasy kit, and further processed in the Illumina TruSeq Stranded mRNA library prep kit with appropriate RNA spike-in to mitigate experimental errors. All RNA-seq samples were analyzed by Tapestation to assess quality, and a Qubit Fluorometer to measure concentration. All RNA seq libraries were sequenced on the Illumina Next-seq 500 with 75 bp read single-end sequencing. For fresh frozen primary samples, 15-25 mg fresh frozen tissue samples were harvested on ice, and washed with cold PBS. Samples were immediately resuspended in cold Qiagen proprietary RLT buffer. The tissue was initially grinded with in a 1.5mL eppendorf tube with a small pestle, and further homogenized using a QiaShredder. Remaining protocol was followed as done with RNA-seq for the cell cultures. RNA-seq libraries were prepared with Illumina's TruSeq standard mRNA Sample Prep Kit using standard protocols. Cut&Tag protocol: CUT&Tag was carried out according to Epicypher's protocol in 8-strip PCR tube with slight modifications. Concanavalin A coated magnetic beads were activated with Bead Activation Buffer containing 20 mM HEPES, pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2; beads were kept at room temperature until used. 100,000 cells were trypsinized, washed with cold PBS to remove trypsin. Cells were lysed sing cold Nuclear Extraction Buffer containing 20 mM HEPES–KOH, pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% Glycerol supplemented with fresh 0.5 mM Spermidine, and cOmplete, Mini, EDTA-free Protease Inhibitor (Roche). Nuclei samples were incubated with activated Concanavalin A beads at room temperature for 10 min. The nuclei-Concanavalin A bead complex was resuspended in Antibody 150 Buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, supplemented with fresh 0.5 mM Spermidine, Protease Inhibitor (Roche), 0.01% Digitonin, and 500 ng of primary antibody. Following overnight incubation at 4°C, primary antibody was removed, and beads were washed once with Digitonin 150 Buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, supplemented with fresh 0.5 mM Spermidine, Protease Inhibitor (Roche), and 0.01% Digitonin. Secondary antibody were added, and incubated with nuclei-Concanavalin A bead complex for 1 hr at room temperature. Nuclei-Concanavalin A bead complex were washed with Digitonin 150 Buffer twice before resuspension in 50uL cold Digitonin300 Buffer containing 20 mM HEPES, pH 7.5, 300 mM NaCl, supplemented with fresh 0.5 mM Spermidine, Protease Inhibitor (Roche), and 0.01% Digitonin. 2.0 µL CUTANA pAG-Tn5 (Epicypher) was added to each sample, and in incubated on a nutator for 1 hr at room temperature. Following incubation, beads were washed with cold Digitonin300 Buffer. Targeted chromatin tagmentation and library amplification were carried out exactly according to Epicypher's protocol.