GSM7106094: KO PH6h rep2; Mus musculus; RNA-Seq
Liver tissue was excised from mice and was immediately submerged in RNA later solution. For mRNA profiling, total RNA was isolated from pooled livers from the control and MeCP2-KO mice using the TRIzol® reagent as the manufacturer instructed. The quality of the total RNA was verified by an Agilent 2100 Bioanalyzer profile. The Illumina adapters used during the library construction were removed from the reads using Trimmomatic. In order to reduce the impact of lower quality reads on the alignment, all reads were trimmed to 60 bp using the FASTX Toolkit, resulting in a Phred-Quality-Score greater than. The reads generated for each RNA sample were analyzed and compared using an Ergatis-based RNA-Seq analysis pipeline where sequencing reads were aligned and annotated to the UCSC mouse reference genome (mm10) from Ensembl using TopHat v-2.0.8,and the number of reads that aligned to the predicted coding regions were determined using HTSeq. Bedtools was used to count the reads mapping to exons according to Ensembl gene annotations. Read counts per million mapped reads values (CPM) or reads per kilobase of transcripts per million mapped reads (RPKM) were calculated and used for downstream analyses. 5′-3′ exonic coverage was calculated using CollectRnaSeqMetrics component of picard-tools, and duplication rate was calculated using Estimate Library Complexity of Picard-tools. Total RNA was extracted using Trizol (Invitrogen, CA, USA), then incubated with RNase-free DNase I (Takara Bio, China) for 30 min at 37 °C to remove residual DNA. Quantity and quality of total RNA were assessed using NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA) and RNase free agarose gel electrophoresis. mRNA was purified using oligo-dT beads (Qiagen). Fragmentation was carried out by adding fragmentation buffer. First-strand cDNA was synthesized using random hexamer-primed reverse transcription. Second-strand cDNA was subsequently synthesized using DNA Polymerase I and RNase H. To select cDNA fragments at a preferential length about 150 bp to 200 bp, fragments were purified using a QIAquick PCR extraction kit. After washed with EB buffer, the gathered fragments were ligated to sequencing adapters. Then, PCR was performed to construct the final cDNA library