GSM3638973
GSM3638973: silenced_RNAPII_ChIP-seq_rep2; Rattus norvegicus; ChIP-Seq
ChIP-seq was essentially performed as described (Heinz et al., 2018), with modifications. Briefly, neuronal maintenance media was aspirated, 2 mM Disuccinimidyl glutarate (DSG; ThermoFisher Scientific Cat#20593) / PBS for 30 minutes at room temp with gentle agitation, followed by an additional 15 minutes with 16% PFA added to 1% final. The reactions were quenched by adding 2.625 M glycine to 125 mM final, incubated for 5 min, then 20% bovine serum albumin (BSA; Fisher Bioreagents Cat#BP9706-100) added to 1% final, the mixture of fixatives aspirated and replaced with ice-cold 0.5% BSA / PBS,  cells collected using a cell scraper into a chilled 15 mL conical tube, and centrifuged at 5000g for 5 min at 4°C. Cells were resuspended in ice-cold LB3 lysis buffer containg the following (in mM) (10 Tris / HCl pH 7.5, 100 NaCl, 0.5 EGTA, 0.1% deoxcycholate, 0.5% sarkosyl, 1× protease inhibitor cocktail (Sigma Aldrich Cat# P8340) and chromatin was sheared to an average DNA size of 300–500 bp by administering 7 pulses of 10 sec duration at 13 W power output with 30 sec pause on wet ice using a Misonix 3000 sonicator. The lysate was diluted in 1.1-fold (LB3) with ice-cold 10% Triton X-100. One percent of the lysate was removed for whole cells inputs. For each immunoprecipitation, aliquots of lysate equivalent to 3ₓ106 cells for transcription factor  and RNAPII antibodies, or 7.5ₓ105 cells for histone modification antibodies, 20 μl of Dynabeads Protein A (for rabbit polyclonal antibodies) or Dynabeads protein G (for murine monoclonal antibodies) and 2 μg of the indicated antibody were combined, and rotated overnight at 8 RPM and 4°C. The next day, beads were collected on a magnet and washed 3x each with wash buffer I (WB1; in mM: 10 Tris / HCl pH 7.5, 150 NaCl, 1% Triton X-100, 0.1% SDS, 2 EDTA), wash buffer III (WB3; in mM: 10 Tris / HCl pH 7.5, 250 LiCl, 1 EDTA, 1% Triton X-100, 0.7% Deoxycholate) and 2x with ice-cold TET (in mM: 10 Tris / HCl pH 7.5, 1 EDTA, 0.025% Tween-20). Beads were then resuspended in 20µL cold TT (in mM: 10 Tris / HCl pH 7.5, 0.025% Tween-20). Libraries were prepared with NEBNext Ultra II DNA library prep kit reagents according to the manufacturer's protocol on the beads suspended in TT, with reagent volumes reduced by half. ChIP samples were indexed by ligation to NEXTflex DNA Barcodes. DNA was eluted and crosslinks reversed by adding 4 μl 10% SDS, 4.5 μl 5 M NaCl, 3 μl EDTA, 1 μl proteinase K (20 mg / ml), 20 μl water, incubating for 1 h at 55°C, then 30 min to overnight at 65°C. DNA was cleaned up by adding 2 μl SpeedBeads 3 EDAC (Sigma Aldrich Cat#GE65152105050250) in 61 μl of 20% PEG 8000 / 1.5 M NaCl, mixing and incubating for 10 minutes at RT. SpeedBeads were collected on a magnet, washed twice by adding 150 μl 80 % EtOH for 30 seconds each, collecting beads and aspirating the supernatant. After air-drying the SpeedBeads, DNA was eluted in 25 μl TT and the DNA contained in the eluate was amplified for 12 cycles in 50 μl PCR reactions using NEBNext Ultra II Q5 PCR master mix (New England BioLabs Cat#M0544S) and 0.5 μM each of primers Solexa 1GA and Solexa 1GB (See Key Resources table). Libraries were cleaned up as above by adding 36.5 μl 20% PEG 8000 / 2.5 M NaCl and 2 μl Speedbeads, two washes with 150 μl 80% EtOH for 30 sec each, air-drying beads and eluting the DNA into 20 μl TT.
GEO
GSM3638973
GEO Accession
GSM3638973