title: GSM6634754: HC4; Homo; RNA-Seq
library_name: GSM6634754
library_construction_protocol: RNA was extracted with TRIzoL (Ambion #15596026, Carlsbad, USA). Library preparation and Illumina sequencing (pair-end sequencing of 151bp) were done at the Genomics Core of the Centre for PanorOmic Sciences in LKS Faculty of Medicine of The University of Hong Kong. cDNA libraries were prepared by KAPA mRNA HyperPrep Kit KR1352-v3.16 (Roche #08098123702, Basel, Switzerland). One microgram of total RNA was used as starting material. Manufacturer's protocol was followed. In brief, Poly-A containing mRNA was collected by using poly-T oligo-attached magnetic beads. The purified mRNA was fragmented to 200 – 300 bp by incubating at 94oC for 6 min in the presence of magnesium ions. The fragmented mRNA was then applied as template to synthesize the first-strand cDNA by using random hexamer-primer and reverse transcriptase. In the second strand cDNA synthesis, the mRNA template was removed and a replacement strand was generated to form the blunt-end double-stranded (ds) cDNA. The ds-cDNA underwent 3' adenylation and ligation with xGen® Dual Index UMI Adapters (Integrated DNA Technologies, Iowa, USA). The adaptor-ligated libraries were enriched by 10 cycles of polymerase chain reaction (PCR). The libraries were denatured and diluted to optimal concentration. Illumina NovaSeq 6000 was used for pair-end 151bp sequencing.