title: GSM2474143: ADC_input; Mus musculus; ChIP-Seq
library_construction_protocol: Lung tumors were pulverized, cross-linked with 1% formaldehyde in PBS for 10 min at RT, washed in 5mg/ml BSA in PBS and then in just cold PBS, re-suspended in lysis buffer [50mM Tris-HCl, pH 8.1, 10mM EDTA, 1% SDS, 1X complete protease inhibitors (Roche)], and sonicated with the Covaris E210 or S2 sonicator to obtain chromatin fragment lengths of 200-to-1500bp judged by Bioanalyzer DNA High sensitivity kit (Agilent). Fragmented chromatin was diluted in IP buffer [20mM Tris-HCl pH 8.1, 150mM NaCl, 2mM EDTA, 1% Triton X-100] and incubated overnight at 4ºC with Protein G magnetic beads (Dynabeads: Life Technologies) that had been pre-incubated with antibodies. Immunoprecipitates were washed six times with wash buffer [50mM HEPES pH 7.6, 0.5M LiCl, 1mM EDTA, 0.7% Na deoxycholate, 1% NP-40] and twice with TE buffer. Immunoprecipitated (or no IP input) DNA was treated with RNase A and Proteinase K on the beads, recovered in 1% SDS and 0.1M NaHCO3 over a period of 5 hours at 65ºC, column purified with DNA clean and concentrator -25 (Zymo Research). After a sonication to enrich DNA fragment lengths between 100-300bp, 5 to 10ng of DNA were used for the library construction using NEBNext Ultra DNA Library Prep Kit (NEB E7370).  Sequencing was performed on a NextSeq (Illumina) for 38 nucleotides from paired ends according to manufacturer’s instructions.
attributes.tag: GEO Accession
attributes.value: GSM2474143