alias: GSM2051558
title: GSM2051558: RNA_Seq_E14_2i_24h_2; Mus musculus; RNA-Seq
library_construction_protocol: RNASeq: RNA was used for library preparation using TruSeq RNA Library Prep Kit v2 (Illumina) following the manufacturer’s instructions. Libraries were indexed either using Illumina Indexes and 50 bp single-end sequencing was performed on Illumina HiSeq 2000 instruments using TruSeq reagents (Illumina, San Diego, CA, USA), according to manufacturer’s instructions. Genomic DNA and total RNA were isolated from the samples using the Beckman Coulter's Agencourt RNAdvance Cell v2 kit following the manufacture’s instructions. RNASeq: RNA was used for library preparation using TruSeq RNA Library Prep Kit v2 (Illumina) following the manufacturer’s instructions. Libraries were indexed either using Illumina Indexes and 50 bp single-end sequencing was performed on Illumina HiSeq 2000 instruments using TruSeq reagents (Illumina, San Diego, CA, USA), according to manufacturer’s instructions. RRBS-Seq was performed as described previously (Smallwood et al, 2011) ChIP-Seq: Cells were cross-linked with 1% methanol-free formaldehyde in fresh medium for 10 minutes. Cross-linking reaction was quenched with glycine at a final concentration of 0.2M. Cells were then washed twice with ice-cold PBS and harvested. Cell pellets were then resuspended in LB1 buffer (50 mM Hepes–KOH, pH 7.5; 140 mM NaCl; 1 mM EDTA;10% Glycerol; 0.5% NP-40 or Igepal CA-630; 0.25% Triton X-100, protease inhibitors) for 10 minutes at 4°C, pelleted and resuspended in LB2 buffer (10 mM Tris–HCL, pH8.0; 200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA, protease inhibitors) for 10 minutes at 4°C. Cells were pelleted, resuspended in LB3 buffer (10 mM Tris–HCl, pH 8; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% Na–Deoxycholate; 0.5% N-lauroylsarcosine, protease inhibitors) and sonicated using Misonix Sonicator 3000. After addition of Triton-X to a final concentration of 1%, the lysate was centrifuged at 20,000g for 10 minutes to pellet debris. Sonicate chromatin was then added to the bead-antibody complexes and incubated overnight at 4°C. Bead-antibody were prepared as follows: Protein G-coupled Dynabeads were incubated overnight with primary antibodies (anti-H3K9me2; Abcam ab1220) in PBS added with 5 mg/ml BSA. Beads were washed extensively with RIPA buffer (50mM HEPES pH 7.6, 1mM EDTA, 0.7% Na deoxycholate, 1% NP-40, 0.5M LiCL), once with 1x TE buffer and eluted in 200ul of buffer containing 1% SDS and 0.1M NaHCO3. Cross-linking was reversed by incubation at 65°C overnight, followed by RNase A treatment at 37°C for 1 hour and Proteinase K treatment at 55°C for 2 hours. DNA was then extracted using Beckman Coulter's Agencourt RNAdvance Cell v2 kit following the manufacture’s instructions. Eluted DNA was then used to perform ChIP-Seq library preparation using the MicroPlex Library Preparation Kit v2 (Diagenode) ChIP-BS-Seq: Cells were cross-linked with 1% methanol-free formaldehyde in fresh medium for 10 minutes. Cross-linking reaction was quenched with glycine at a final concentration of 0.2M. Cells were then washed twice with ice-cold PBS and harvested. Cell pellets were then resuspended in LB1 buffer (50 mM Hepes–KOH, pH 7.5; 140 mM NaCl; 1 mM EDTA;10% Glycerol; 0.5% NP-40 or Igepal CA-630; 0.25% Triton X-100, protease inhibitors) for 10 minutes at 4°C, pelleted and resuspended in LB2 buffer (10 mM Tris–HCL, pH8.0; 200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA, protease inhibitors) for 10 minutes at 4°C. Cells were pelleted, resuspended in LB3 buffer (10 mM Tris–HCl, pH 8; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% Na–Deoxycholate; 0.5% N-lauroylsarcosine, protease inhibitors) and sonicated using Misonix Sonicator 3000. After addition of Triton-X to a final concentration of 1%, the lysate was centrifuged at 20,000g for 10 minutes to pellet debris. Sonicate chromatin was then added to the bead-antibody complexes and incubated overnight at 4°C. Bead-antibody were prepared as follows: Protein G-coupled Dynabeads were incubated overnight with primary antibodies (anti-H3K9me2; Abcam ab1220) in PBS added with 5 mg/ml BSA. Beads were washed extensively with RIPA buffer (50mM HEPES pH 7.6, 1mM EDTA, 0.7% Na deoxycholate, 1% NP-40, 0.5M LiCL), once with 1x TE buffer and eluted in 200ul of buffer containing 1% SDS and 0.1M NaHCO3. Cross-linking was reversed by incubation at 65°C overnight, followed by RNase A treatment at 37°C for 1 hour and Proteinase K treatment at 55°C for 2 hours. DNA was then extracted using Beckman Coulter's Agencourt RNAdvance Cell v2 kit following the manufacture’s instructions. Eluted DNA was then treated for WGBS as described before
identifiers.namespace: GEO
identifiers.id: GSM2051558