GSM5570785: RNAseq_IAA_N1; Homo sapiens; RNA-Seq
RNA-seq: 5 x 10^6 cells per sample were lysed using TRIZOL. The RNA was phase separated with chloroform, precipitated with isopropyl alcohol, and DNase treated . ChIP-seq:ChIP-seq: Cells were resuspended in PBS at 4x106 cells/ml, and crosslinked with 1% methanol free formaldehyde for 10 min at room temperature before quenching with 125 mM Glycine. Cells were pelleted, washed twice with 1.5 ml PBS, and lysed in lysis buffer (50 mM Tris pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton, 0.1% SDS and protease inhibitor cocktail) at a concentration of 80x106 cells per ml. Aliquots of 300 ul were sonicated in a Bioruptor for 20 min and debris was removed by centrifugation. 100 ul of Chromatin was diluted to 1 ml using lysis buffer lacking SDS and incubated overnight at 4C with the appropriate antibody or control IgG. Antibodies were captured on 15 ul bed volume Protein A agarose beads and immune complexes were washed with 1 ml Low Salt Buffer (20 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton), 1 ml Hi Salt Wash Buffer (20 mM Tris pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton), 1 ml LiCl Wash Buffer (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% Triton) and twice with 1 ml of TE. Samples were decrosslinked overnight in 100 ul TE containing 20 ug of Proteinase K (PK) at 65C then brought up to 500 ul with water and incubated at 95C for 20 minutes to inactive PK. PRO-seq: Nuclei from 30 x 10^6 Ramos cells per sample were isolated in buffer using dounce homogenization then flash frozen and kept at -80C until run on reaction. Primary transcripts were eluted using TRIZOL extractions and dynabeads, which isolate primary transcripts by binding the biotin incorperated during the run-on. RNA-seq: Library preparation with rRNA depletion performed by Vanderbilt Technologies for Advanced Genomics (VANTAGE) PRO-seq: RNA was used in a reverse transcriptase reaction to generate a cDNA library. Libraries were amplified based on the a PCR cycle number determined from a test analysis of a portion of sample. Library amplification was performed with Illumina-based index primers. ChIP-seq: ChIP-seq: After de-crosslinking, samples were purified using the MinElute PCR Purification Kit (Qiagen). The NEBNext Ultra II DNA Library Prep Kit for Illumina was used to prepare unique dual indexed libraries from the entire eluate following the manufacture's protocol. RNA-seq, ChIP-seq, PRO-seq