GSM4721536: Pol2_Mock; Homo sapiens; ChIP-Seq
RNA-Seq: Total RNA was extracted using NucleoSpin® RNA Plus kit (Macherey-nagel) according to the manufacture's instruction, including an on-column genomic DNA digestion step.  Quality and quantity of RNA was checked with Qubit and Bioanalyzer. Ribosomal RNA (rRNA) depletion was performed using NEBNext® rRNA Depletion Kit (Human/Mouse/Rat). cDNA libraries were prepared by NEBNext® Ultra II Directional RNA Library Prep Kit. Five hundred nanogram of total RNA was used as starting material for ribosomal RNA depletion. Cytoplasmic rRNA and mitrochondrial rRNA were firstly hybridized to ssDNA capture probes and then digested by Ribonuclease H. After that, the residual ssDNA probes were digested by DNase I. The rRNA-depleted RNA was then purified by Agencourt® RNAClean™ XP beads. The purified rRNA-depleted RNA was fragmented to 200 – 500 bp by incubating at 94oC for 10 min in the presence of magnesium ions. The fragmented rRNA-depleted RNA was then applied as template to synthesize the first-strand cDNA by using random hexamer-primer and reverse transcriptase. In the second strand cDNA synthesis, the mRNA template was removed and a replacement strand was generated to form the blunt-end double-stranded (ds) cDNA. The ds cDNA underwent, 3' adenylation and indexed adaptor ligation, followed by treatment with User Enzyme. The adaptor-ligated libraries were enriched and indexed by 8 cycles of polymerase chain reaction (PCR). The libraries were denatured and diluted to optimal concentration. Illumina NovaSeq 6000 was used for Pair-End 151bp sequencing. ChIP-Seq: Infected cells were fixed with 1.5% formaldehyde for 12 minutes. Cells were subsequently lyzed with lysis buffer (20mM Tris-HCL pH8.0, 85 mM KCL, 0.5% NP-40) on ice for 20 min. Cell nuclei were isolated by centrifuge and resuspended in 1% SDS lysis buffer (50 mM Tris-HCL pH8.0, 5 mM EDTA, 1% SDS). Sonication was then performed (Bioruptor, 8 cycles of 30 sec on and 30 sec off, three times). Samples were then diluted and split into four fractions for each antibody pulldown(1- Millipore, 05-623; 2- Abcam, ab5131; 3- Abcam, ab5095) and plus alpha (4- 10% input) (4 ug antibody per assay). Pulled down experiments used Protein G-beads (Invitrogen, 10004D) followed by washing and elution. Reverse-crosslinked was done overnight (with salt and RNase) and incubated with Proteinase K and finally column-purified for library prep. Library preparation utilized the NEBNext Ultra II DNA Library Prep Kit (NEB, E7645), following manufacturers intructions. For ChIP-seq, total RNA was Size-selected before PCR (targeting 200 bp) with SPRI beads (Beckman, B23317). PCR consisted of three cycles for inputs and 11-13 cycles for ChIP-ed samples. Library preparation and Illumina sequencing (Pair-End sequencing of 151bp) were done at The University of Hong Kong, LKS Faculty of Medicine, Centre for PanorOmic Sciences (CPOS), Genomics Core.
GEO Accession
GSM4721536