Total RNA was extracted from whole blood using PAXgene Blood RNA Kit (PreAnalytiX), followed by treatment with GLOBINclear™ kit (ThermoFisher Scientific) to remove unwanted globin mRNA. The remaining RNA product was used to prepare cDNA library using Illumina TruSeq Stranded mRNA library preparation kit (Illumina). RNAseq was performed by Beijing Genomics Institute using the Illumina Hiseq2000 with 75 base-pair (bp) paired-end reads to a depth of at least 30 million reads per sample.
RNAseq output was processed as previously described [76 (link)]. Read pairs were pre-processed to adjust base calls with phred <5 to ‘N’ and to remove read pairs where either end had fewer than 30 unambiguous base calls. This method indirectly removes read pairs containing mostly adaptor sequences. Read pairs were aligned to the human genome (hg19) using STAR (v2.3.1d) [77 (link)]. Gene counts were tabulated using htseq (v0.6.0) with the intersection-strict setting turned on and Ensembl gene annotations (GRCh37.74) used to map genomic locations to gene identifiers [78 (link)]. The edgeR (v3.36.0) package function, cpm, was used to calculate TMM-normalized counts-per-million (CPM) expression matrices [79 (link)].
RNAseq output was processed as previously described [76 (link)]. Read pairs were pre-processed to adjust base calls with phred <5 to ‘N’ and to remove read pairs where either end had fewer than 30 unambiguous base calls. This method indirectly removes read pairs containing mostly adaptor sequences. Read pairs were aligned to the human genome (hg19) using STAR (v2.3.1d) [77 (link)]. Gene counts were tabulated using htseq (v0.6.0) with the intersection-strict setting turned on and Ensembl gene annotations (GRCh37.74) used to map genomic locations to gene identifiers [78 (link)]. The edgeR (v3.36.0) package function, cpm, was used to calculate TMM-normalized counts-per-million (CPM) expression matrices [79 (link)].
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