GSM1643961: p65 ChIP-Seq IL1b Rep2; Homo sapiens; ChIP-Seq
ChIP-Seq: Sequencing libraries were prepared from collected DNA by blunting, A-tailing, adapter ligation as previously described (Heinz, S. et al. Molecular Cell 38, 576-589 (2010)) using barcoded adapters (NextFlex, Bioo Scientific). Libraries were PCR-amplified for 12-15 cycles, size selected by gel extraction and sequenced on a HiSeq 2000 (Illumina) for 51 cycles. RNA-Seq: Enriched mRNA was hydrolyzed with Fragmentation Buffer (Ambion) for 10 min at 70°C and re-buffered to 10 mM Tris pH 7.4 using P-30 size exclusion columns (Bio-Rad). RNA was then de-capped for 2 h at 37°C with 0.5 µl (10 U/µl) TAP (Epicentre) in 20 µl TAP buffer containing 1 U/µl SUPERase-IN. Samples were 3’ dephosphorylated for 50’ at 37°C with 1 µl PNK (Enzymatics), 0.5 µl 10x TAP buffer, 1.5 µl water, 0.5 ul 0.25 M MgCl2 (3 mM free Mg2+ final), 0.5 µl 10 mM ATP (0.2 µM final to protect PNK). Subsequently, RNA was 5’-phosphorylated for 60 min at 37°C by adding 2 µl (10 U/µl) PNK, 10 ul 10x T4 DNA ligase buffer and 63 µl water. RNA was extracted with Trizol LS, precipitated in the presence of Glycoblue (Ambion), and dissolved in 4.5 µl water. 0.5 µl 9 µM of a 5’-adenylated sRNA3'MPX adapter /5Phos/AG ATC GGA AGA GCA CAC GTC TGA /3AmMO/ (IDT, desalted; adenylated with Mth ligase (NEB) according to the manufacturer’s instructions, phenol-chloroform/chloroform-extracted, ethanol-precipitated with glycogen and dissolved in water at 9 µM) were heat-denatured together with the RNA for 2 minutes at 70°C, and ligated with 100 U truncated T4RNA ligase 2 K227Q (NEB) in 10 µl 1x T4 RNA ligase buffer without ATP, containing 10 U SUPERase-In and 15% PEG8000 for 2 hours at 16°C. To reduce adapter dimer formation, 0.5 µl 10 µM MPX_ RT primer 5’-GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T-3’ (IDT, desalted) was added and annealed to the ligation product by incubating at 75°C for 2 minutes, then 37°C for 30 minutes, then 25°C for 15 minutes. Finally, 0.5 µl 5 µM hybrid DNA/RNA sRNA5'h adapter 5’-GTT CAG AGT TCT ACA rGrUrC rCrGrA rCrGrA rUrC-3’ (IDT) were ligated to previously capped RNA 5’ ends by adding 2 µl T4 RNA ligase buffer, 6 µl 50% PEG8000 (15% final), 1 µl 10 mM ATP, 9.5 µl water and 0.5 µl (5 U) T4 RNA ligase 1 for 90 minutes at 20°C. To 15 µl ligation reaction, an additional 0.5 µl 10 µM MPX_ RT primer were added, reactions were denatured at to 70°C for 1 minute, then placed on ice. RNA was reverse-transcribed by adding 3 µl 10x first strand buffer, 4.5 µl water, 1.5 µl 10 mM dNTP, 3 µl 0.1 M DTT, 1.5 µl RNaseOUT and 1 µl Superscript III reverse transcriptase (Invitrogen), and incubating for 30 minutes at at 50°C. Complementary DNA was isolated by adding 35 µl AMPure XL beads (Beckman), binding and washing according to manufacturer’s instructions and dissolved in 40 µl TET. Libraries were PCR-amplified for 13 cycles with 0.75 µM primers oNTI201 (5'-AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GAC G-3' and TruSeq-compatible indexed primers (e.g. 5’-CAA GCA GAA GAC GGC ATA CGA GAT iii iii GTG ACT GGA GTT CAG ACG TGT GCT CTT-3’ (desalted, IDT, i signifies index nucleotides) using Phusion Hot Start II (Thermo Scientific) in HF buffer containing 0.5 M betaine (98°C, 30s/12x(98°C, 10s/57°C, 25s/72°C, 20s)/ 72°C, 1min/4°C, ?), 175-225 bp fragments were size-selected on 10% PAGE gels and sequenced for 51 cycles on a HiSeq 2000 sequencer (Illumina) with small RNA sequencing primer 5’-CGA CAG GTT CAG AGT TCT ACA GTC CGA CGA TC-3’ and TruSeq Index sequencing primer (Illumina). Gro-Seq: Nuclei were extracted from 8-12 million cells grown on 10 cm plates and nuclear run-on reactions were performed for 5 minutes at 30°C in the presence of Sarkosyl and BrUTP. RNA was extracted with Trizol, DNase-treated, base-hydrolyzed and dephosphorylated with PNK. BrUTP-labeled run-on RNA was immunopurified with anti-BrdUTP-coated agarose beads and precipitated overnight. Poly(A)-tailing was followed by cDNA synthesis using complementary poly(T)-primers. Excess oligo was removed by Exonuclease I and cDNA fragments were purified on a denaturing 10% polyacrylamide TBE-urea gel. The recovered cDNA was circularized, linearized, amplified for 10-16 cycles. The final product was run on 10% TBE gel, gel purified and cleaned-up using ChIP DNA clean & Concentrator Kit. The final libraries were sequenced using an Illumina HiSeq 2000.