GSM9359750: CVG-1-d-CBP1, biol rep2; Homo sapiens; RNA-Seq
Total RNA was extracted with Trizol (Invitrogen) and quantified using Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA).   ERCC RNA Spike-In Mix (Cat: #4456740) from ThermoFisher Scientific, was added to normalized total RNA prior to library preparation following manufacturer's protocol. RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads.  Enriched mRNAs were fragmented for 15 minutes at 94 °C. Converted the RNA molecules into complementary DNA (cDNA) and incorporated UMIs during reverse transcription. cDNA fragments were end repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were clustered on a flowcell. After clustering, the flowcell was loaded on the Illumina NovaSeq instrument according to manufacturer's instructions.  The samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the Control software. Raw sequence data (.bcl files) generated by the sequencer were converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.