GSM6754676: NL_B_RNAseq_Rep2; Homo sapiens; RNA-Seq
We isolated total RNA from using the mirVana miRNA Isolation Kit (Thermo Fisher, AM1560) according to the manufacturer's protocol. We used 100 ng of isolated RNA for RNA-seq library preparation using TruSeq Stranded Total RNA Library Prep Gold (Illumina, 20020598) according to the manufacturer's instruction. In brief, we removed rRNA from the input RNA, followed by double stranded cDNA preparation using 0.8 U of SuperScript II RT (Thermo Fisher, 4376600) and A-tailing end repair. We ligated cDNA to TruSeq RNA Single Indexes Set A (Illumina, 20020492) to enable multiplex sequencing, followed by one round of of size selection (selecting for 300 bp) and bead clean-up : 42.5 uL of sample was purified with 42 uL of Agencourt AMPure XP beads (Beckman Coulter, A63881), then, 50 uL sample was cleaned with 50 uL Agencourt AMPure XP beads (Beckman Coulter, A63881). We amplified the purified samples by 15 PCR cycles and further purified using Agencourt AMPure XP beads (Beckman Coulter, A63881). We assessed library quality and quantities using the Agilent DNA 1000 reagent kit (Agilent, 5067–1504) on the Agilent Bioanalyzer 2100 (Agilent, 5067–4626) and Qubit high sensitivity RNA assay kit (Thermo Fisher, Q32852), respectively before sequencing on NextSeq500 (Illumina). We ligated cDNA to TruSeq RNA Single Indexes Set A (Illumina, 20020492) to enable multiplex sequencing, followed by one round of of size selection (selecting for 300 bp) and bead clean-up : 42.5 uL of sample was purified with 42 uL of Agencourt AMPure XP beads (Beckman Coulter, A63881), then, 50 uL sample was cleaned with 50 uL Agencourt AMPure XP beads (Beckman Coulter, A63881). We amplified the purified samples by 15 PCR cycles and further purified using Agencourt AMPure XP beads (Beckman Coulter, A63881). We assessed library quality and quantities using the Agilent DNA 1000 reagent kit (Agilent, 5067–1504) on the Agilent Bioanalyzer 2100 (Agilent, 5067–4626) and Qubit high sensitivity RNA assay kit (Thermo Fisher, Q32852), respectively before sequencing on NextSeq500 (Illumina).