title: GSM1285796: Tcf1_CAT; Mus musculus; ChIP-Seq
library_construction_protocol: 1e08 WT or CAT thymocytes were fixed in 3ml of RPMI containing 1% formaldehyde for 10 min at room temperature. The fixative was quenched with glycine, samples were washed twice with ice cold PBS and cells were lysed in RIPA buffer for 10 min on ice. Samples were then sonicated using Diagnode's Bioruptor water bath sonicator (desired fragment size=200bp, conditions vary). Lysates were cleared by centrifugation and incubated overnight with Tcf-1 antibodies [a kind gift of Hiroshi Kawamoto (Riken Yokohama Institute, Yokohama, Japan)] or H3K4me3 antibodies coupled to Protein G Dynabeads (Life Technologies). Immunoprecipitation and input samples were de-cross-linked overnight at 65 °C in elution buffer [50 mM Tris·HCl (pH 8)/10 mM EDTA/1% SDS/ 0.3 M NaCl]. DNA was extracted with phenol/chloroform, quantified, and quality-checked for enrichment of known target regions of Tcf-1 by real-time PCR. ChIP-Seq libraries were prepared from 10 ng of immunoprecipitated material according to an Illumina ChIP DNA library preparation kit. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer II following the manufacturer's protocols.
attributes.tag: GEO Accession
attributes.value: GSM1285796