title: GSM4151539: SBMA_Bioreplicate_2; Homo sapiens; ChIP-Seq
library_construction_protocol: iMNs were first treated with 1% formaldehyde for 10 min n 37o C, followed by 0.125 M glycine in PBS. iMNs were then resuspended in RIPA buffer supplemented with EDTA-free protease inhibitor (ROCHE). After, cells were sonicated using TC12x24 tube on Covaris S2, duty cycle 20%, intensity 5, cycle/burst 200, 60s x 30 cycles. For immunoprecipitation, Dynabeads Protein A was incubated with 10 μg ChIP-antibody for 45 min at RT on a shaker. The pre-cleared chromatin was transferred into the antibody-bound beads and incubated on a tube rotator overnight in 4o C. Following overnight incubation, samples were washed for 10 min with rotation at 4o C with 2x RIPA buffer, 2x RIPA + 0.3 M NaCl, 2x LiCl buffer. After the last wash beads were re-suspended with TE (pH8) +10% SDS supplemented with 20 mg/mL proteinase K and incubated at 65o C for 4 hours in a shaker. The supernatant was collected using a magnet and DNA was purified using phenol/chloroform/isoamyl alcohol. The DNA concentration was measured using Pico-Green dsDNA assay kit (ThermoFisher). Libraries were constructed from 10 ng of ChIP DNA using used the Illumina Nextera XT kit. These libraries were then purified twice using Ampure XP PCR Purification Beads (Agencourt). Following purification, libraries were pooled then quantitated by qPCR. The balance of the pool was subsequently checked by performing a MiSeq run of 25 cycles plus index read using a MiSeq Nano kit, version 2. The percentage of each library in the pool was determined from the demultiplexing and was used to optimize the pool balance prior to final paired-end (2x50bp) sequencing using an Illumina HiSeq 2500.
attributes.tag: GEO Accession
attributes.value: GSM4151539