title: GSM1838871: Pancreas SA1-R1_ChIP-seq; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP was performed in tissues (cerebral cortex and pancreas, in the case of 8-10 week old C57Bl/6 mice, and whole brain in the case of E17.5 embryos) from wildtype, SA1 heterozygous and SA1-null mice with custom made rabbit polyclonal antibodies against SA1 and SMC1 and RNApolIISer2P (AB5095, Abcam) as described (Cuadrado et al. 2010). Mice were housed in a pathogen-free animal facility following the animal care standards of the institution. Fresh tissues collected in 6 MW plates containing cold PBS with 1 mM PMSF and protease inhibitor cocktail (Roche) were immediately minced to get small fragments of about 1 mm diameter. Tissue pieces (pooled from three individuals) were centrifuged at 2000 rpm for 2 minutes at 4ºC, resuspended in 1 ml of fixing solution (1% formaldehyde, 50 mM HEPES-KOH, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) and incubated 20 min at room temperature in a rotating wheel. Cross-linking was stopped by adding 1/20 volume of 2.5M Glycine for 5 minutes at RT. Tissue pieces were washed twice with cold PBS, recovered by centrifugation at 3000 rpm for 7 minutes at 4ºC, resuspended in 2 ml lysis buffer (1% SDS, 10mM EDTA, 50 mM Tris-HCl pH 8.1; 2x107 cells per ml) and sonicated in Covaris system (shearing time 30 min, 20% duty cycle, intensity 10, 200 cycles per burst and 30 seconds per cycle). DNA samples were provided by the user. 15 ng of ChIP DNA as quantitated by fluorometry was used. Electrophoretic fraction of 100-200bp was processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters as in Illumina's "TruSeq DNA Sample Preparation Guide" (part # 15005180 Rev. C). Adapter-ligated library was completed by limited-cycle PCR with Illumina PE primers (11 cycles). The resulting purified DNA library was applied to an Illumina flow cell for cluster generation (TruSeq cluster generation kit v5) and sequenced on the Genome Analyzer IIx with SBS TruSeq v5 reagents by following manufacturer's protocols.
attributes.tag: GEO Accession
attributes.value: GSM1838871