GSM8671582: siSCR H4s, replicate 6, sequencing batch 3; Homo sapiens; RNA-Seq
Total RNA was extracted from dry cell pellets following the manufacturer's protocol for the Monarch Total RNA Miniprep Kit (New England Bioscience). All centrifugation steps were performed at 16,000 g and at room temperature, and the DNase 1 treatment step was completed. RNA was eluted in 30 μL water and concentration was measured using a NanoDrop 1 (Thermofisher). RNA extracted for sequencing was diluted to 50 ng/μL in water. Bulk RNA-sequencing libraries were constructed and sequenced by the UCL Genomics Facility. Total RNA (500 ng) was used as input for the construction of complementary DNA (cDNA) libraries with the KAPA messenger RNA (mRNA) HyperPrep kit (Roche), as per manufacturer's instructions. First, magnetic oligodT beads were used to capture mRNA, which was then fragmented using heat and magnesium. Random priming was used for first strand cDNA synthesis, followed by combined second strand synthesis and A-tailing. Adapter ligation was then performed, followed by strand-specific library amplification (further details of which can be found here: https://elabdoc-prod.roche.com). xGen Dual Index UMI Adapters (Integrated DNA Technologies, Inc.) were used to minimise read misassignment in later de-multiplexing of samples. Libraries were multiplexed on the NovaSeq SP v1.5 for paired-end 100 bp sequencing on the NovaSeq 6000 Sequencing System (Illumina), aiming for a mean read depth of 66 million paired-end reads per sample. Sequenced reads were de-multiplexed and BCL Convert software (Illumina) was used to generate FASTQ files.