title: GSM4420283: CREB3L2-CREB3L2 homodimers HA immunoprecipitation; Homo sapiens; ChIP-Seq
library_construction_protocol: Twenty-four hours post-transfection, we proceeded by crosslinking protein-DNA contacts with 1% (v/v) formaldehyde (#28908, Thermo Fisher) for 10 minutes at room temperature. After quenching cross-linking reaction with glycine, cells were washed twice with ice-cold PBS, harvested by scrapping in PBS with protease inhibitors, and centrifuged at 2,000 g and 4ºC, as per manufacturer's instructions (SimpleChIP Plus Kit [#9005], Cell Signaling Technology). Chromatin fragments (mainly 1-3 nucleosomes in size) were obtained by incubation with micrococcal nuclease (3.5 ul [equivalent to 7,000 gel units] in 200 ul; M0247S, NEB) for 45 minutes in a 37ºC-water bath. Nuclear membranes were subsequently broken up by three rounds of 20-second, 15% amplitude pulses using a Sonic Dismembrator Model 500 (Fisher Scientific), and lysates clarified by centrifugation. Adequate digestion was assessed by agarose gel electrophoresis. For each condition, digested chromatin was split into two tubes and immunoprecipitated overnight at 4ºC with end-over-end rotation using pre-washed magnetic beads conjugated with anti-HA- or anti-V5 antibodies (anti-HA beads: PI88836, Thermo Fisher; anti-V5 beads: NC0777490, MBL International); 35 ul of anti-HA beads and 25 ul of anti-V5 beads were utilized per immunoprecipitation. Beads were captured on a magnetic stand and washed with low- and high-salt buffers, as directed. Elution was performed at 65ºC and 1,200 rpm for 30 minutes using a thermomixer, protein-DNA cross-links reversed by treatment with Proteinase K for 2 hours at 65ºC, and DNA column-purified. A representative 2% input sample was prepared by combining chromatin from the different backgrounds. ChIP-seq library preparation and sequencing reactions were conducted at GENEWIZ, Inc., as described above. A total of 24 samples were submitted for analysis, comprising parallel HA and V5 immunoprecipitations, from 2 independent replicates. The sequencing libraries were multiplexed and clustered on two lanes of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq instrument according to manufacturer's instructions (Illumina, San Diego, CA, USA). Sequencing was performed using a 2x150 Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mis-match was allowed for index sequence identification.
attributes.tag: GEO Accession
attributes.value: GSM4420283