title: GSM1946487: tissue RNA-Seq WT control #1; Homo sapiens; RNA-Seq
library_construction_protocol: The frozen tissues were cut and divided into 50 mg aliquots and stored in tubes at -80°C. The frozen tissues were homogenized on ice for 15–30 seconds in 500 μL 1X PBS using a tissue grinder (ACTGene, Piscataway, NJ). Tissue homogenates or cultured cells were cross-linked to final 1% formaldehyde for 10 min and quenched with 125 mM glycine for 5 min at room temperature and washed once with TBS. The pellets were resuspended in cell lysis buffer (10 mM Tris-HCl, pH7.5, 10 mM NaCl, 0.5% NP-40) and incubated on ice for 10 min. The lysates were divided into two aliquots and washed with MNase digestion buffer (20 mM Tris-HCl, pH7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2). After resuspending in 500 μL MNase digestion buffer containing a proteinase inhibitor cocktails (Sigma, St. Louis, MO), the lysates were incubated in the presence of 1,000 units of MNase (NEB, Ipswich, MA, Cat.# M0247S) at 37 °C for 20 min with continuous mixing in thermal mixer (Fisher Scientific, Pittsburgh, PA). After adding the same volume of sonication buffer (100 mM Tris-HCl, pH8.1, 20 mM EDTA, 200 mM NaCl, 2% Triton X-100, 0.2% sodium deoxycholate), the lysates were sonicated for 15 min (30 sec on / 30 sec off) using Bioruptor Twin (UCD-400) (Diagenode, Inc., Denville, NJ) and centrifuged at 21,130 x g for 10 min. The cleared supernatant equivalent to 10–20 mg of tissue or 1–2x106 cells was incubated with 2 ug of rabbit polyclonal anti-H3K36me3 antibody (Active Motif, Carlsbad, CA, Cat.# 61101) or 1.5 ug of rabbit monoclonal anti-H3K36me2 antibody (Cell Signaling Technology, Danvers, MA, Cat.#2901) on rocker overnight. After adding 30 μL protein G-magnetic beads (Life technologies, Carlsbad, CA), the reactions were incubated for 3 hours. The beads were extensively washed with ChIP buffer (50 mM Tris-HCl, pH8.1, 10 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), high salt buffer (50 mM Tris-HCl, pH8.1, 10 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), LiCl2 buffer (10 mM Tris-HCl, pH8.0,0.25 M LiCl2, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA), and TE buffer. Bound chromatins were eluted and reverse-crosslinked at 65°C overnight. DNAs were purified using Min-Elute PCR purification kit (Qiagen, Valencia, CA) after the treatment of RNase A and proteinase K.For RNA-seq, total RNA was extracted using the miRNeasy Mini kit (Qiagen, Valencia, CA). ChIP-seq libraries were prepared from 10 ng ChIP and input DNA using the Ovation ultralow DR Multiplex kit (NuGEN, San Carlos, CA). The ChIP-seq libraries were sequenced to 51 base pairs from both ends on an Illumina HiSeq 2000 instrument in the Mayo Clinic Center for Individualized Medicine Medical Genomics Facility.RNA-seq libraries were prepared with ovation RNA-seq system v2 kit (NuGEN) according to the manufacture’s instruction, and were sequenced on an Illumina HiSeq 2000 instrument in the Mayo Clinic Center for Individualized Medicine Medical Genomics Facility.
attributes.tag: GEO Accession
attributes.value: GSM1946487