GSM9288857: 20230427_MACR065_MOLM13_shSMARCD2_V5-SMARCD2_H3K27ac; Homo sapiens; OTHER
CUT&RUN: Cells were pelleted by centrifugation (300 rcf, 5 minutes, room temperature) and were washed with PBS containing 200mM Glycine. Cells were resuspended with 100ul/sample of Nuclear Extraction Buffer containing 20 mM HEPES pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% Glycerol supplemented with fresh 0.5 mM Spermidine and 1X protease inhibitor (Sigma Aldrich, Cat# 11836170001) for 2 min on ice. Nuclei were pelleted by centrifugation (600 rcf, 3 minutes, 4C), and resuspended in 100ul/sample cold Wash150 Buffer containing 20 mM HEPES pH 7.9, 150 mM NaCl, 1% Triton X-100, 0.05% SDS supplemented with fresh 0.5 mM Spermidine and 1X protease inhibitor (Sigma Aldrich, Cat# 11836170001) and incubated with activated ConA beads at room temperature for 15 minutes. Nuclei-ConA bead complexes were resuspended in 50 ul/sample cold Antibody Buffer containing 20 mM HEPES pH 7.9, 150 mM NaCl, 1% Triton X-100, 0.05% SDS, 2mM EDTA supplemented with fresh 0.5 mM Spermidine, 1X protease inhibitor (Sigma Aldrich, Cat# 11836170001), 0.01% Digitonin, and 0.5 ug primary antibody/sample, and incubated overnight nutating at 4C. Following removal of supernatant, ConA-nuclei complexes were washed twice with Digitonin Buffer containing 20 mM HEPES pH 7.9, 150 mM NaCl, 1% Triton X-100, 0.05% SDS supplemented with fresh 0.5 mM Spermidine, 1X protease inhibitor (Sigma Aldrich, Cat# 11836170001), and 0.01% Digitonin. Samples were then resuspended in 50ul Digitonin Buffer and 2 ul of CUTANA pAG-MNase (Epicypher, Cat#: 15-1116) each and incubated at room temperature for 10 minutes. Samples were washed twice with Digitonin Buffer and resuspended in 50ul Digitonin Buffer and 1ul of 100mM CaCl2 per sample. Following incubation nutating at 4C for 2 hours, samples received 33ul of Stop Buffer containing 340 mM NaCl, 20 mM EDTA, 4 mM EGTA supplemented with fresh 50 ug/mL RNase A, 50 ug/ml Glycogen and incubated at 37C for 1 hour to release MNase digested DNA fragments. Supernatant was collected into fresh tubes and supplemented with 0.8uL 10% SDS and 1uL of 20mg/mL Proteinase K for overnight incubation at 55C. DNA was purified with Ampure XP (Beckman Coulter, Cat#: A63881) beads and 80% EtOH washes. DNA was eluted with 25uL of 0.1X TE buffer and transferred to new tubes. DNA fragments were prepared for next generation sequencing with CUTANA CUT&RUN Library Prep Kit (Epicypher, Cat#: 14-1001) according to manufacturer protocol. ATAC-seq: Nuclei were resuspended in transposition mix (1X TD Buffer, 2.5 ul TDE1 Nextera Tn5 Transposase (Illumina)) and incubated with mild rotation for 30 min at 37°C. Tagmented DNA was purified using MinElute PCR cleanup kit (Qiagen). RNA-seq: 2 X 106 live cells as were washed with PBS and RNA was extracted using QIAshredder and RNeasy Mini Kit (Qiagen) per the manufacturer's instructions. CUT&TAG: Cells were processed for Cut & Tag as previously published and according to manufacturer's CUTANA protocol (Epicypher) with the use of BioMag Plus Concanavalin A beads (Polysciences), CUTANA pAG-Tn5 (Epicypher), primary antibodies, and Guinea Pig anti-Rabbit IgG (Heavy & Light chain) antibody (Antibodies-Online). Tn5 transposed fragments were amplified between 14-18 cycles using NEB Next HF 2X PCR Master Mix (NEB) and custom primers originally designed for ATAC-seq library amplification to allow for multiplexing of 50+ samples. Amplified DNA was purified using 0.75X AMPpure Beads (Beckman Coulter) to remove primer-dimers. CUT&RUN: Concanavalin A (ConA) beads (Polysciences, Cat#: 86057-10) were activated with Bead Activation Buffer containing 20 mM HEPES pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2; beads were stored on ice until used. 500,000 cells per sample were harvested and resuspended in media at 1 million cells per mL. Cells were lightly crosslinked with 16% paraformaldehyde at a final concentration of 0.1% at room temperature for 2 minutes. Crosslinking was quenched with 2.5M Glycine at a final concentration of 200mM at room temperature for 5 minutes.  Cells were pelleted by centrifugation (300 rcf, 5 minutes, room temperature) and were washed with PBS containing 200mM Glycine. Cells were resuspended with 100ul/sample of Nuclear Extraction Buffer containing 20 mM HEPES pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% Glycerol supplemented with fresh 0.5 mM Spermidine and 1X protease inhibitor (Sigma Aldrich, Cat# 11836170001) for 2 min on ice. Nuclei were pelleted by centrifugation (600 rcf, 3 minutes, 4C), and resuspended in 100ul/sample cold Wash150 Buffer containing 20 mM HEPES pH 7.9, 150 mM NaCl, 1% Triton X-100, 0.05% SDS supplemented with fresh 0.5 mM Spermidine and 1X protease inhibitor (Sigma Aldrich, Cat# 11836170001) and incubated with activated ConA beads at room temperature for 15 minutes. Nuclei-ConA bead complexes were resuspended in 50 ul/sample cold Antibody Buffer containing 20 mM HEPES pH 7.9, 150 mM NaCl, 1% Triton X-100, 0.05% SDS, 2mM EDTA supplemented with fresh 0.5 mM Spermidine, 1X protease inhibitor (Sigma Aldrich, Cat# 11836170001), 0.01% Digitonin, and 0.5 ug primary antibody/sample, and incubated overnight nutating at 4C. Following removal of supernatant, ConA-nuclei complexes were washed twice with Digitonin Buffer containing 20 mM HEPES pH 7.9, 150 mM NaCl, 1% Triton X-100, 0.05% SDS supplemented with fresh 0.5 mM Spermidine, 1X protease inhibitor (Sigma Aldrich, Cat# 11836170001), and 0.01% Digitonin. Samples were then resuspended in 50ul Digitonin Buffer and 2 ul of CUTANA pAG-MNase (Epicypher, Cat#: 15-1116) each and incubated at room temperature for 10 minutes. Samples were washed twice with Digitonin Buffer and resuspended in 50ul Digitonin Buffer and 1ul of 100mM CaCl2 per sample. Following incubation nutating at 4C for 2 hours, samples received 33ul of Stop Buffer containing 340 mM NaCl, 20 mM EDTA, 4 mM EGTA supplemented with fresh 50 ug/mL RNase A, 50 ug/ml Glycogen and incubated at 37C for 1 hour to release MNase digested DNA fragments. Supernatant was collected into fresh tubes and supplemented with 0.8uL 10% SDS and 1uL of 20mg/mL Proteinase K for overnight incubation at 55C. DNA was purified with Ampure XP (Beckman Coulter, Cat#: A63881) beads and 80% EtOH washes. DNA was eluted with 25uL of 0.1X TE buffer and transferred to new tubes. DNA fragments were prepared for next generation sequencing with CUTANA CUT&RUN Library Prep Kit (Epicypher, Cat#: 14-1001) according to manufacturer protocol. CUT&RUN experiments were performed in one replicate but with antibodies for different mSWI/SNF subunits. Overlap between distinct subunit peaks were used for high confidence in results. ATAC-seq: ATAC-seq was performed as previously described with 100,000 cells per sample. Cells were dissociated and counted, as described above, 7-days post transduction with lentivirus. Cells were washed with cold PBS, resuspend in lysis buffer (10mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL) and centrifuged at 500 g for 10 min at 4°C. Nuclei were resuspended in transposition mix (1X TD Buffer, 2.5 ul TDE1 Nextera Tn5 Transposase (Illumina)) and incubated with mild rotation for 30 min at 37°C. Tagmented DNA was purified using MinElute PCR cleanup kit (Qiagen). Samples were amplified using 10 total PCR amplification cycles with custom PCR Primers using NEBNext HF PCR Master Mix (NEB). Samples were purified with 1.2X Agencourt AMPure XP beads (Beckman Coulter) and size distribution was determined using D5000 HS reagents and screentapes (Agilent) on 2200 TapeStation Instrument (Agilent). Samples were pooled in equimolar amounts and sequenced on Illumina Next-seq 500 with NextSeq 500/550 High Output Kit V2 75 cycles (Illumina) using 37bp paired-end sequencing parameters. ATAC-Seq experiments were performed in two replicates. RNA-seq: Cells were dissociated and counted, as described above, 7-days post transduction with lentivirus. 2 X 106 live cells as were washed with PBS and RNA was extracted using QIAshredder and RNeasy Mini Kit (Qiagen) per the manufacturer's instructions. 1 µg of total RNA was used as input for library preparation and processed per the manufacturer's instructions using NEBNext Poly(A) mRNA magnetic isolation module (NEB) followed by the NEBNext Ultra II Directional RNA library prep kit for Illumina (NEB) with NEBNext Multiplex Oligos for Illumina (NEB). Samples were collected in biological replicate. ERCC synthetic RNA spike-in control (Thermo Fisher Scientific) was added at the beginning of library preparation per manufacturer's recommendations to normalize samples across conditions. Libraries were quantified using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and size distribution was verified using Tapestation D1000 HS reagents and screentapes (Agilent). Equimolar amounts of libraries were pooled, denatured, and sequenced on Illumina Next-seq 500 with NextSeq 500/550 High Output Kit V2 75 cycles (Illumina) according to standard Illumina protocols with single-end sequencing parameters. RNA-Seq were performed in two biological replicates. CUT&TAG: CUT&TAG was performed following published protocols (Epicypher). Cells were dissociated from culture plates using TrypLE Express (Thermo Fisher Scientific) and washed once with PBS pH 7.4 (Gibco). Cells were counted with Countess II (Invitrogen). 200,000-500,000 live cells were used per epitope as determined by Trypan Blue (Thermo Fisher Scientific) exclusion. Cells were processed for Cut & Tag as previously published and according to manufacturer's CUTANA protocol (Epicypher) with the use of BioMag Plus Concanavalin A beads (Polysciences), CUTANA pAG-Tn5 (Epicypher), primary antibodies, and Guinea Pig anti-Rabbit IgG (Heavy & Light chain) antibody (Antibodies-Online). Tn5 transposed fragments were amplified between 14-18 cycles using NEB Next HF 2X PCR Master Mix (NEB) and custom primers originally designed for ATAC-seq library amplification to allow for multiplexing of 50+ samples. Amplified DNA was purified using 0.75X AMPpure Beads (Beckman Coulter) to remove primer-dimers. Libraries were quantified and QC'd as above. Samples were sequenced by the Dana Farber Cancer Institute Molecular Biology Core Facility using paired-end read parameters on an Illumina Nextseq 500 (Illumina) using Illumina NextSeq 500/550 High Output Kit V2 75 cycles (Illumina).