title: GSM2109707: G1_WT1; Mus musculus; RNA-Seq
library_construction_protocol: For RNA-seq Total RNA was extracted using the RNeasy kit (Qiagen) For ChIP-seq assays were performed according to the Millipore protocol. Cells were fixed using 1% formaldehyde, harvested, resuspended in ChIP lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1) and sonicated using Bioruptor Pico (Diagenode) to generate fragments of 150 to 300 bp. Soluble chromatin was diluted 8 fold in ChIP RIPA buffer (10mMTris–HCl, pH 7.5, 140mMNaCl, 1mMEDTA, 0.5mMEGTA,1%Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate) and incubated with Dynabeads Protein A (Invitrogen) coupled to H3K36me2 antibody ab9049 from Abcam. After incubation, the immunocomplexes were washed sequentially with Low Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 150mM NaCl), High Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl), LiCl Wash Buffer (0.25M LiCl, 1% NP40, 1% deoxycholate, 1mM EDTA, 10mM Tris-HCl, pH 8.1) and TE. Immunocomplexes were eluted in ChIP elution buffer (1%SDS, 0.1M NaHCO3) and the crosslinking was reverted overnight at 65 ºC. Samples were treated with Proteinase K and RNase A and DNA was extracted using the QIAGEN PCR purification kit. Purified chromatin was used for library construction. For RNA-seq 1 µg of total RNA sample was used . PolyA+ fraction was purified and randomly fragmented, converted to double stranded cDNA and processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters as in Illumina's "TruSeq Stranded mRNA Sample Preparation Part # 15031047 Rev. D" kit (this kit incorporates dUTP during 2nd strand cDNA synthesis, which implies that only the cDNA strand generated during 1st strand synthesis is eventually sequenced). Adapter-ligated library was completed by PCR with Illumina PE primers (8 cycles). The resulting purified cDNA library was applied to an Illumina flow cell for cluster generation and sequenced on an Illumina instrument (see below) by following manufacturer's protocols. For ChIP-seq the amount of DNA used was 15ng from each sample. Samples were processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters with "NEXTflex ChIP Sequencing kit" from Bioo Scientific (part # 5143). Adapter-ligated libraries were completed by limited-cycle PCR (13 cycles) and extracted with a double double-sided SPRI size selection. Libraries were applied to an Illumina flow cell for cluster generation and sequenced on an Illumina instrument (see below) by following manufacturer's protocols.
attributes.tag: GEO Accession
attributes.value: GSM2109707