GSM1424440: 14D Conceptus input; Ovis aries; ChIP-Seq
Conceptus tissue samples (~100 mg) were submersed in 1% formaldehyde in PBS, cut into small pieces, and incubated at room temperature for 15 min. Fixation was stopped by the addition of 0.125 M glycine. Tissue pieces were then treated with a TissueTearor (BioSpecProducts, Bartlesville, OK, USA) 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 (Misonix, 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 decrosslinking, followed by phenol/chloroform extraction and ethanol precipitation. Purified DNA was quantified on a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA). Extrapolation to the original chromatin volume allowed quantization of the total chromatin yield. For each ChIP reaction, 30 ng of chromatin was precleared with protein A agarose beads (Invitrogen, Carlsbad, CA, USA). Immunoprecipitation was performed using 4 μg of rabbit anti PPARG polyclonal IgG (# sc-7196; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Following overnight incubation at 4°C, protein A agarose was added, and incubation at 4°C continued for another 3 h. Immune complexes were washed 2 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. Input and ChIP DNA were amplified using an Illumina ChIP-Seq DNA Sample Prep Kit (Illumina, San Diego, CA, USA). 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 bp on a 2% agarose gel. After a PCR amplification(30 s at 98°C; followed by 10 s at 98°C, 30 s at 65°C, and 30 sat 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 reverse transcription quantitative PCR (RT-qPCR) at the same specific genomic regions as the original ChIP DNA to assess quality of the amplification reactions. The DNA libraries were sent to Illumina Sequencing Services for sequencing on an Illumina HiSeq 2000.