GSM2481926: EAAE V; Mus musculus; ChIP-Seq
Uteri were submersed in PBS + 1% formaldehyde, cut into small (~1 mm3) pieces with a razor blade and incubed at room temperature for 15 minutes. Fixation was stopped by the addition of 0.125 M glycine (final). The tissue pieces were then treated with a TissueTearer and finally spun down and washed 2x in PBS. Chromatin was isolated from the sample by adding 5-10 ml lysis buffer containing PIPES, Igepal, PMSF and Protease Inhibitor Cocktail, followed by disruption with a Dounce homogenizer. Samples were pelleted by centrifugation and resuspended in buffer containing Na deoxycholate, SDS, and Triton X-100. Lysates were sonicated using a Misonix Sonicator 3000 equipped with a microtip in order to shear the DNA to an average length of 300-500 bp. Lysates were cleared by centrifugation and the chromatin suspensions were transferred to new tubes and stored at -80 C. Prior to use in ChIP, protein A agarose beads (Invitrogen) were preblocked using blocking proteins and nucleic acids for 3 hr. For each ChIP reaction, an aliquot of chromatin (20-30 ug) was precleared with 30 ul preblocked protein A agarose beads for 1-2 hr. ChIP reactions were set up using precleared chromatin and antibody Eralpha (sc-542, Lot# D1310) in a buffer containing Na deoxycholate and incubated overnight at 4 C. Preblocked protein A agarose beads were added and incubation at 4 C was continued for another 3 hr. Agarose beads containing the immune complexes were washed two times each with a series of buffers consisting of the deoxycholate sonication buffer, high salt buffer, LiCl buffer, and TE buffer. An SDS-containing buffer was added to elute the immune complexes from the beads, and the eluates were subjected to RNase treatment at 37 C for 20 min and proteinase K treatment at 37 C for 3 hr. Crosslinks were reversed by incubation overnight at 65 C, and ChIP DNAs were purified by phenolchloroform extraction and ethanol precipitation. Quality of ChIP enrichment was assayed by qPCR using primers against candidate EREs in the Igf1 and Stat5a promoters. Input DNA was queried at the same sites in parallel ChIP DNA was amplified by following the Illumina ChIP-Seq DNA Sample Prep Kit protocol. In brief, DNA ends were polished and 5’-phosphorylated using T4 DNA polymerase, Klenow polymerase and T4 polynucleotide kinase. After addition of 3’-A to the ends using Klenow fragment (3’-5’ exo minus), Illumina genomic adapters were ligated and the sample was sizefractionated (200-250 bp) on a 2% agarose gel. After a final PCR amplification step (18 cycles,Phusion polymerase), the resulting DNA libraries were quantified and tested by QPCR at the same specific genomic regions as the original ChIP DNA to assess quality of the amplification reactions. DNA libraries were sequenced on a Genome Analyzer II.
GEO
GSM2481926
GEO Accession
GSM2481926