title: GSM1602748: RNA-Seq NMDA; Mus musculus; RNA-Seq
library_construction_protocol: Chromatin immunoprecipitation was performed using a ChIP assay kit (Millipore) as described by the manufacturer. Briefly, cultured primary neurons (1 x 10^7 cells) were crosslinked by treatment with formaldehyde (1% final) for 10 minutes at 37°C, washed twice with ice-cold PBS, scraped, and resuspended in lysis buffer containing 50mM Tris pH 8, 10mM EDTA, and 1% SDS. Cells were sonicated using a bioruptor (medium power, 30 seconds on/30 seconds off – 20 cycles).  Sheared chromatin was immunoprecipitated variously with antibodies against either γH2AX (Abcam – ab2893), or Topo IIβ (Abcam – ab58442), and using salmon sperm DNA-protein A-agarose beads. Crosslinked proteins were then eluted from the beads in a solution containing 100mM NaHCO3 and 1% SDS. Crosslinks were reversed by incubation at 65°C overnight, following which DNA was extracted using phenol/chloroform/isoamyl alcohol. For RNA-seq, total RNA was extracted from cultured primary neurons using the Qiagen RNeasy Plus Universal Kit (Qiagen) and by following the manufacturer's recommended protocol. Sequencing libraries were prepared from ~1-5 ng ChIP (or input) DNA as described previously (Ernst et al., 2011). Gel electrophoresis was used to retain library fragments between 300 and 600 bp.  Prior to sequencing, libraries were quantified using Quibit (Invitrogen) and quality-controlled using Agilent's Bioanalyzer.  36bp single-end sequencing was performed using the Illumina HiSeq 2000 platform according to standard operating procedures. Sequencing reads were mapped to mouse genome assembly (mm9) using BWA aligner (samse option). For RNA-seq, Illumina TruSeq Total RNA Sample Prep Kits (Illmina) was used for library preparation. The bar-coded libraries were quantified before equal mixing for sequencing on the Illumina Hi-Seq 2000 system. The raw fastq data files of paired-end reads were collected for analysis.
attributes.tag: GEO Accession
attributes.value: GSM1602748