title: GSM3212887: lgs303260_Input Chromatin ChIP-seq control; Homo sapiens; ChIP-Seq
library_construction_protocol: For ChIP-seq, we crosslinked 10 million Epstein-Barr Virus (EBV)-transformed B cells with 1% formaldehyde for 5 minutes followed by careful cell lysis using the truChIP Chromatin Shearing Kit (Covaris).  Nuclei were isolated and fragmented using a Covaris E220e sonicator   with the following operating conditions: Peak Incident Power: 140 Watts; duty cycle: 5%; cycles per burst: 200; treatment time: 15 minutes; setpoint temperature: 6°C).  Crosslinked protein/DNA was immunoprecipitated using H3K27ac (rabbit polyclonal, Abcam #ab4729, 20ug/ml)  or H3K4me1 (rabbit polyclonal, Abcam #ab8895, 20ug/ml) antibodies on Protein A + G immunomagnetic beads.  Anti-acetyl histone H3 antibodies and isotype matched IgG antibodies were included as positive and negative controls, respectively.  An aliquot of the fragmented chromatin, not subjected to immunoprecipitation, was also used as an input control.  Chromatin crosslinks were reversed, then protein was digested with proteinase K and DNA fragments were purified using Ampure XP beads (Beckman Coulter).  For HiChIP-sequencing, We crosslinked 10 million EBV-transformed B cells using 1% formaldehyde for 10 minutes at room temperature.  Intact nuclei were isolated and digested using the MboI restriction enzyme for 4 hours at 37C.  DNA fragment ends were filled and labeled with dCTP, dGTP, dTTP and biotin-dATP. Proximity ligation was performed at room temperature overnight.  Following the in situ ligation, we fragmented the ligated chromatin as previously described64.  Crosslinked protein/DNA was immunoprecipitated using antibodies to H3K27ac (rabbit polyclonal, Abcam #ab4729, 20ug/ml) and CTCF (rabbit mAb, clone D31H2, Cell Signaling #3418, 20ug/ml) and then purified by Protein A+G immunomagnetic beads.  The amount of MboI enzyme, antibody, and beads are determined by the number of cells in each sample64. After immunoprecipitation, DNA was eluted from the beads by incubating at 65C for 4 hours with 5% Proteinase K. DNA was purified by Zymo DNA Clean & Concentrator Column and quantified by Qubit High Sensitivity Assay Kit.  RNA was isolated from each LCL using TRIzol. DNA and RNA libraries were prepared for sequencing using standard Illumina protocols. DNA libraries were sequenced on the Illumina NextSeq 500 using 75bp single-end reads.  RNA libraries were sequenced on the Illumina HiSeq 3000 using 75bp paired-end reads with 10 samples per lane, yielding 30 million reads per sample.
attributes.tag: GEO Accession
attributes.value: GSM3212887