title: GSM7051762: MOLM13 OE RBM5 cDNA targeted cells rep1 [MOLM13-R5_OE_1]; Homo sapiens; RNA-Seq
library_name: GSM7051762
library_construction_protocol: RNA samples of 3 biological replicates were extracted from cultured cells, using TRIZOL (Ambion by Life technologies, 343911), following the manufacturer's instructions. About 200 ng total RNA was treated using Kapa rRNA depletion reagents to remove ribosomal RNA, then converted into cDNA libraries using Kapa RNA HyperPrep Kit with RiboErase (HMR). After end repair, dA-tailing, and adapter ligation, each cDNA library was purified and enriched by 11 cycles of PCR amplification. All RNA-seq libraries underwent 101-cycle paired-end sequencing on the Illumina Novaseq system. Quality control filtering and adapter trimming of fastq files was performed with trim_galore. Paired-end reads were mapped by STAR(v2.7.1a) using parameters "-c -p 4 --outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJstitchMismatchNmax 5 -1 5 5 --alignSJDBoverhangMin 10 --outFilterMismatchNmax 999 --outFilterMismatchNoverReadLmax 0.04 --alignIntronMin 20 --alignIntronMax 100000 --alignMatesGapMax 100000 --outSAMmapqUnique 60 --outSAMmultNmax 1 --outSAMstrandField intronMotif --outSAMattributes NH HI AS nM NM MD --outSAMunmapped Within --outSAMtype BAM SortedByCoordinate --outReadsUnmapped None --chimJunctionOverhangMin 12 --chimSegmentReadGapMax 3 --chimMultimapNmax 10 --chimMultimapScoreRange 10 --chimNonchimScoreDropMin 10 --chimOutJunctionFormat 1 --quantMode TranscriptomeSAM GeneCounts --twopassMode Basic --peOverlapNbasesMin 12 --peOverlapMMp 0.1 --outWigType wiggle --outWigStrand Stranded --outWigNorm RPM". Read duplication was marked with MarkDuplicates from GATK (v4.1.2.0). RSEM was used to quantify read counts per gene. Differentially expressed genes were identified using the generated count data described above and DESeq2 (version 1.14.0).