title: GSM5456029: ChIP_gw_CTCF_salt_rep1; Homo sapiens; ChIP-Seq
library_construction_protocol: C-TALE was performed as described previously (Golov AK, Ulianov SV, Luzhin AV, Kalabusheva EP, Kantidze OL, Flyamer IM, Razin SV, Gavrilov AA. C-TALE, a new cost-effective method for targeted enrichment of Hi-C/3C-seq libraries. Methods. 2020 Jan). For ChIP-seq experiments, fixed chromatin pellets were resuspended in 600 uL of ice cold RIPA buffer supplemented with protease inhibitors. Chromatin was sheared on ice with a VirSonic 100 cell disrupter with 10 30-sec pulses on “15” power setting, separated with 3 min periods of recovery. Input DNA was isolated from 1/10 aliquots of sonicated samples. Sheared chromatin from approximately 1 mln of cells and 1 ug of antibodies (against either CTCF, Smc3, or Rad21) were used in each immunoprecipitation reaction. Chromatin immunoprecipitation was performed as described in Abcam X-ChIP manual (https://www.abcam.com/protocols/cross-linking-chromatin-immunoprecipitation-x-chip-protocol) with minor modifications. 25 ul of protein A/G magnetic beads (Thermo Scientific, 26162) was used per reaction instead of agarose beads, thus a magnet was used instead of a centrifuge to reclaim beads. Beads were blocked with 1% BSA solution in RIPA overnight, while chromatin was incubated with antibodies. BSA-blocked beads were mixed with antibody-bound chromatin for 6 h. Immunoprecipitated DNA was separated from beads by overnight treatment with proteinase K (Thermo Scientific, EO0491) in PBS supplemented with 1% SDS at 65C. Solution was then cleared from beads with a magnet and DNA was isolated with standard phenol-chloroform extraction and ethanol precipitation. Sequencing libraries from 3C-seq material, immunoprecipitated DNA and ChIP inputs were generated as described previously (Golov AK, Ulianov SV, Luzhin AV, Kalabusheva EP, Kantidze OL, Flyamer IM, Razin SV, Gavrilov AA. C-TALE, a new cost-effective method for targeted enrichment of Hi-C/3C-seq libraries. Methods. 2020 Jan).