title: GSM3399583: LC_3_0h_Me3; Homo sapiens; ChIP-Seq
library_construction_protocol: for ChIP-seq: Purified migratory LCs were fixed with 1% formaldehyde for 15 min and the reaction quenched with 0.125 M glycine. Chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 100–200 bp (Covaris). Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat to remove crosslinks, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. Genomic DNA regions of interest were isolated using appropriate antibodies. Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation (at 37 °C for 1 h, followed by 4 h at 65 °C) overnight at 65 °C, and ChIP DNA was purified by phenol–chloroform extraction and ethanol precipitation.  For RNA-seq: RNA was isolated using RNeasy mini kit (Qiagen) as per the manufacturer's protocol. RNA concentration and integrity was determined with an Agilent Bioanalyser (Agilent Technologies, Santa Clara, CA). All the samples had a RNA integrity number of 7.0 or above and were taken forward for labelling. for Chip-seq: Illumina sequencing libraries were prepared from the ChIP and input DNAs by the standard consecutive enzymatic steps of end- polishing, dA-addition, and adaptor ligation using TruplexTM –FD prep kit (Rubicon Genomics, USA). After a final PCR amplification step, the resulting DNA libraries were quantified using Qubit (Life technologies, USA) and 75 nucleotide single-end reads were sequenced Illumina HiSeq2500 in the DNA sequencing core of the Cincinnati Children's Hospital Medical Center.  For RNA-seq: RNA-seq libraries were generated from 300 ng total RNA with an RNA Sample Prep Kit (Illumina) according to a standard protocol. The libraries were sequenced with Illumina HiSeq2500 in the DNA sequencing core of the Cincinnati Children's Hospital Medical Center. Each sample was used to generate 2 × 107 reads with 75–base pair paired-end sequencing.
attributes.tag: GEO Accession
attributes.value: GSM3399583