title: GSM4087828: input-control-ChIP-seq; Mus musculus; ChIP-Seq
library_construction_protocol: For each ChIP experiment, 107 mouse ESCs were used. Cells were washed with PBS and cross-linked with 1% formaldehyde (Fisher Scientific) in PBS for 10 min at room temperature. The reaction was quenched with 125 mM glycine. Fixed cells were washed with cold PBS, scraped, centrifuged, resuspended in cold lysis buffer (15 mM HEPES (pH 7.5), 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.5% N‑lauroylsarcosine, 0.1% sodium deoxycholate, 0.1% SDS), incubated for 10 min on ice and sonicated with a Bioruptor Pico for four cycles of 30 s on and 30 s off.  ChIP-seq experiments were performed as previously described in Koenecke et al 2017 "Drosophila poised enhancers are generated during tissue patterning with the help of repression" with 107  mouse ESCs per ChIP. For each ChIP, 5 µg of the following antibodies were used: ɑ-Oct3/4 (Santa Cruz, sc-8628), ɑ-Sox2 (Santa Cruz, sc-17320), or ɑ-Nanog (Santa Cruz, sc-30328). At least two biological replicates were performed for each factor. Single-end sequencing was performed on either an Illumina HiSeq instrument (50 cycles) or NextSeq 500 instrument (75 cycles) according to manufacturer's instructions. Sequencing libraries were prepared for sequencing using standard Illumina protocols The ChIP-nexus procedure and data processing were performed as previously described in Johnston et al 2015 except that the ChIP-nexus adaptor mix contained four fixed barcodes (ACTG, CTGA, GACT, TGAC). For each ChIP, 5 µg antibody was coupled to 50 µl of Dynabeads Protein A or Protein G (Invitrogen). The following antibodies were used: ɑ-Oct3/4 (Santa Cruz, sc-8628), ɑ-Sox2 (Santa Cruz, sc-17320), ɑ-Nanog (Santa Cruz, sc-30328), ɑ-Klf4 (R&D Systems, AF3158), ɑ-Klf4 (Abcam, ab106629), ɑ-Esrrb (Abcam, ab19331), ɑ-Pbx 1/2/3 (Santa Cruz, sc-888), and ɑ-Zic3 (Abcam, ab222124).
attributes.tag: GEO Accession
attributes.value: GSM4087828