title: GSM2811312: LAM_Input  rep2; Mus musculus; ChIP-Seq
library_construction_protocol: Chromatin IP was performed according to a previously published protocol (Schmidt et al., 2009) with a few adjustments. Briefly, DRG pellets (20 mice/sample) were crushed using an automatic pestle and chemically cross-linked with a 1% formaldehyde solution containing 50mM Hepes-KOH pH7.5, 100mM NaCl, 1mM EDTA and 0.5mM EDTA, for 15 minutes at room temperature. Nuclear extracts were then sonicated for 30 min using a Bioruptor (Diagenode) and successful chromatin shearing (200-800 bp) was confirmed by agarose gel analysis. Immunoprecipitation was performed overnight with Protein G Dynabeads (Invitrogen) bound to 20 μg of HDAC3 antibody (Millipore 17-10238). After washing, elution and reverse crosslinking, DNA was treated with RNAse A and Proteinase K and purified using Qiagen PCR purification columns. The concentration of the recovered DNA was quantified with Picogreen assay and the enrichment of the immunoprecipitate calculated with respect to the input and IgG. Library preparation was performed using the NEBNext Ultra DNA Library Prep Kit from Illumina (NEW ENGLAND BioLabs) following the manufacturer's protocol. Briefly, 2 ng of Input and IP DNA were end repaired and adaptor ligated, using a dilution of 1:20 of adaptors. Samples were cleaned to remove unligated adaptors and size selection was performed to select and enrich fragments of 200bp in size. Libraries were amplified by PCR using multiplex oligo following manufacturer's protocol. Prior to sequencing, libraries were run on an Agilent Bioanalyser for size and quality control checking. Sequencing was performed in an Illumina HiSeq2000, generating 50bp single ended reads.
attributes.tag: GEO Accession
attributes.value: GSM2811312