title: GSM3526513: V5Zglp1OE_c8PGCLCs_DoxRA_Input; Mus musculus; ChIP-Seq
library_construction_protocol: The harvested cells were centrifuged at 220×g for 3 min, suspended in 1 mL of PBS and fixed by the addition of 27 µL of 36.5% formalin (Sigma-Aldrich, F8775) for 12 min at room temperature with constant mixing on a tube rotator.  Fixation was terminated by the addition of 110 µL of 1.5 M glycine (Wako, 077-00735).  The fixed cells were washed twice in PBS (Thermo Fisher Scientific, 20012-027) containing 0.1% BSA fraction V and were lysed in 400 µL of SDS-lysis buffer [50 mM Tris-HCl (pH 8.0) (Nacalai Tesque, 35435-11), 1% SDS, 10 mM EDTA, and 1 mM PMSF] on ice for 10 min.  The lysate was split amongst 4 tubes (Diagenode, 315-81401) at 100 µL/tube, and chromatins were sonicated using Picoruptor (Diagenode, 316-81311) at 4 °C for 19 cycles, yielding DNA fragments of 100–300 bp.  The sonicated products from 4 tubes were combined and centrifuged at 16,000×g for 5 min.  The supernatants were diluted in 1 mL ChIP dilution buffer [16.7 mM Tris-HCl (pH 8.0), 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA (Nacalai Tesque, 14347-21) and 167 mM NaCl (Nacalai Tesque, 31334-51)] and divided into 0.1 mL aliquots as input DNA and 1.3 mL aliquots as ChIP samples. For ChIP samples, 15 µg of rabbit polyclonal anti-V5 antibody was reacted with 40 µL of M280 Dynabeads Protein G (Thermo Fisher Scientific, 10003D) at room temperature for 40 min.  ChIP samples were then mixed with the antibody-Dynabeads complexes and incubated at 4 °C for 22 hrs with gentle mixing on a tube rotator.  The chromatin-antibody-Dynabeads complexes were recovered using a DynaMag-2 magnet (Thermo Fisher Scientific, 123.21D) and washed with 200 µL of a low salt buffer [20 mM Tris-HCl (pH 8.0), 0.1% SDS, 1% Triton X-100, 2 mM EDTA, and 150 mM NaCl], a high salt buffer [20 mM Tris-HCl (pH 8.0), 0.1% SDS, 1% Triton X-100, 2 mM EDTA, and 500 mM NaCl], RIPA buffer [10 mM Tris-HCl (pH 8.0), 1% sodium deoxycholate (Nacalai Tesque, 10712-96), 1% NP40 (Nacalai Tesque, 23640-94), 250 mM LiCl (Wako, 244-00461), and 1 mM EDTA], and TE buffer [10 mM Tris-HCl (pH 8.0), and 1 mM EDTA].  After transferring to a new tube, the chromatin was then incubated in 50 µL of elution buffer [0.1 M NaHCO3 (Nacalai Tesque, 31213-15), 1% SDS, 10 mM DTT Nacalai Tesque, 14128-91], and 60 ng/µL tRNA (Roche, 10109517001)] for 15 min at room temperature.  The eluted chromatins were separated from the Dynabeads using DynaMag-2, then reverse-crosslinked by the addition of 8 µL of 2.5 M NaCl and incubated at 65 °C for 16 hrs. Then 2 µL of 0.5 M EDTA, 4 µL of 1 M Tris-HCl (pH 6.5), and 0.2 µL of 20 mg/mL proteinase K (Sigma-Aldrich, P2308) were added to the reaction mix, and the reaction mix was further incubated at 45 °C for 1 hr. The ChIP'd DNAs were purified with Qiaquick PCR purification columns (Qiagen, 28104) using buffer EB (Qiagen, 19086) containing 4 ng/µL of tRNA.  Input chromatins were reverse-crosslinked, treated with proteinase K, and purified in the same way as the ChIP'd samples. Library preparations for the next-generation sequencing were performed as previously described (Mitani, 2017, GSE91038), except that ChIP'd DNAs were not sonicated further by Covaris.  The libraries were sequenced on NextSeq (Illumina) to generate 86 bp single-end reads using a custom read primer (Mitani, 2017, GSE91038).  A total of 8 samples [ESC Dox+RA (ChIP'd), ESC Dox+RA (input), c7 Dox only (ChIP'd), c8 Dox only (ChIP'd), c8 Dox only (input), c7 Dox+RA (ChIP'd), c8 Dox+RA (ChIP'd), and c8 Dox+RA (input)] were sequenced.
attributes.tag: GEO Accession
attributes.value: GSM3526513