GSM4407349: mut6_RNAseq; Mus musculus; RNA-Seq
For ES cells, RNA was extracted from the same number of cells using Qiagen RNeasy mini columns. Synthetic RNAs from the Exteral RNAs Control Consortium (ERCC) were spiked in at known concentrations to the same volume of RNA.    For ChIP in ES cells, 1 million cells were sorted and cross-linked for 10 min a room temperature in 1% formaldehyde/PBS. Cross-links were quenched with glycine (final 125 mM) for 5 min at room temperature. Cell pellets were washed in cold PBS, snap frozen, and stored at -80˚C. All subsequent steps were performed on ice or at 4˚C. Fixed cell pellets were thawed and lysed in 1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8 with protease inhibitors (1x Protease inhibitor cocktail, 1 mM PMSF, 1 mM NaF) for for 30 min. Chromatin was sheared to 200-500 bp fragments on a Covaris S220 with settings PIP 105, duty 2, cpb 200 for 9 min. Chromatin lysates were clarified by centrifugation and diluted 1:10 in dilution buffer (1% Triton X-100, 2 mM EDTA, 167 mM NaCl, 20 mM Tris-HCl pH 8) with protease inhibitors. Inputs were collected at the same time. IPs were performed overnight using 2.5 ug of antibody (CST #9733 H3K27me3) per equivalent of 500,000 cells, rotating at 4˚C. The next day, immunocomplexes were precipitated by incubation with pre-washed Protein A Dynabeads for 2h. Beads were washed 4x10 min in low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl pH 8), 1x10 min in high-salt buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 500 mM NaCl, 20 mM Tris-HCl pH 8), 1x10 min in LiCl buffer (0.25 M LiCl, 0.5% NP-40, 0.5% Na deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8), and 1x fast in TE. ChIP and input samples were eluted in fresh ChIP elution buffer (1% SDS, 50 mM NaHCO3, 50 mM Tris-HCl pH 8, 1 mM EDTA) and treated with RNase A for 1h at 37˚C. Cross-links were reversed by shaking overnight at 65˚C with Proteinase K.    For embryos, RNA was extracted from whole E8.5 embryos cleaned of extraembryonic tissues using Qiagen RNeasy mini columns. For ES cells, RNA-seq libraries were constructed starting from 1 ug of total RNA using the NEBNext Directional II Directional RNA Library Kit for Illumina (NEB #E7760S) and Poly(A) mRNA Magnetic Isolation Module (NEB #E7490S). Libraries were indexed with NEBNext Multiplex Oligos for Illumina and sequenced as a pool.     ChIP-seq libraries were constructed using the NEBNext Ultra II DNA Library Kit for Illumina (NEB #E7645S). Libraries were indexed with NEBNext Multiplex Oligos for Illumina and sequenced as a pool.     For embryos, RNA-seq libraries were constructed starting from 300 ng of total RNA using the NEBNext Directional II Directional RNA Library Kit for Illumina (NEB #E7760S) and Poly(A) mRNA Magnetic Isolation Module (NEB #E7490S). Libraries were indexed with NEBNext Multiplex Oligos for Illumina and sequenced as a pool.