GSM4173689: Crispr TFAM RNAseq rep1; Rattus norvegicus; RNA-Seq
Total RNAs from treatment and control groups were isolated and quality controlled according to the Illumina protocols. A total of 2 μg RNA per sample was used as initial material for library construction. Chromatin immunoprecipitation (ChIP) was performed as described previously (Wang et al., 2⁠ 009b; Wang et al., 2⁠ 008). In brief, cells were crosslinked with 1% formaldehyde for 10 min at room temperature and the reaction was quenched with 125 mM glycine. After chromatin fragmented to 100 to 1000bp by sonication, chromatin templates from 20 million cells were used for ChIP experiment. Samples were immunoprecipitated with 2- 4μg of H3K27ac antibody overnight at 4 °C followed by washing steps. For RNAseq library construction, Poly-A containing mRNA molecules was purified by using poly-T oligo-attached magnetic beads and fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity is achieved by replacing dTTP with dUTP, followed by second strand cDNA synthesis. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter. The products are then purified and enriched with PCR to create the final cDNA library. For ChIPseq library construction, after reverse cross-linking, ChIP-DNA fragments were purified and constructed into libraries by using the ThruPLEX DNA-seq Kit (Rubicon Genomics, Takara, Japan). Amplified libraries around 300 – 500 bp were isolated from agarose gel prior to sequencing.
GEO
GSM4173689
GEO Accession
GSM4173689