GSM1173227: DOPP2.5+3.5_POL2; Mus musculus; ChIP-Seq
Uterine tissue samples (~ 100 mg) were submersed in PBS + 1% formaldehyde, cut into small pieces and incubated at room temperature for 15 minutes.  Fixation was stopped by the addition of 0.125 M glycine.  The tissue pieces were then treated with a TissueTearer and finally spun down and washed twice in PBS.  Chromatin was isolated by disrupting the cells with a Dounce homogenizer.  Lysates were sonicated using a Misonix Sonicator 3000 (Vernon Hills, IL, USA) equipped with a microtip in order to shear the DNA to an average length of 300-500 bp.  Lysates were cleared by centrifugation and stored at -80 C.  Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by phenol/chloroform extraction and ethanol precipitation.  Purified DNA was quantified on a NanoDrop spectrophotometer.  Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield.  For each ChIP reaction, 30 ug of chromatin was precleared with protein A agarose beads (Invitrogen, Carlsbad, CA, USA).  Immunoprecipitation was performed using 4 µg of goat anti-FOXA2 polyclonal IgG (Catalog number sc-6554 from Santa Cruz Biotechnology, Santa Cruz, CA, USA) or 2 µg of rabbit anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) polyclonal antibody (Catalog number ab5095 from Abcam, Cambridge, MA, USA).  Following overnight incubation at 4oC, Protein A agarose was added and incubation at 4oC continued for another 3 h.  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. Immune complexes were eluted from the beads with SDS buffer, and subjected to RNase treatment and proteinase K treatment.  Crosslinks were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.  ChIP and input DNA were amplified using the Illumina ChIP-Seq DNA Sample Prep Kit. Briefly, DNA ends were polished and 5-phosphorylated using T4 DNA polymerase, Klenow polymerase and T4 polynucleotide kinase. After addition of 3’-adenine to blunt ends using Klenow (3’-5’ exo minus), Illumina genomic adapters were ligated and the sample was size fractionated to 200-250 base pairs (bp) on a 2% agarose gel.  After a PCR amplification [30 sec at 98°C; (10 sec at 98°C, 30 sec at 65°C, 30 sec at 72°C) for 18 cycles followed by 5 min at 72°C] with Phusion High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA), the resulting DNA libraries were tested by RT-qPCR at the same specific genomic regions as the original ChIP DNA to assess quality of the amplification reactions.