GSM7882036: 4SD_R1_Illumina; Homo sapiens; RNA-Seq
Cells were harvested by scraping or rinsing in ice cold PBS followed by centrifugation at 270 x g for 10 minutes. Cell pellets were washed twice in ice cold PBS and either used immediately or flash frozen in liquid nitrogen and stored at -80°C. RNA was extracted using TRIzol (Invitrogen) For long read sequencing: RNA was then reverse transcribed with TGIRT-III reverse transcriptase (InGex). Following reverse transcription, RNA was digested with RNase H (New England Biolabs) to remove any bound TGIRT-III and cDNA purified with the Qiagen MinElute kit. To prepare the 5′ adapter, single stranded DNA adapter oligo was treated with Mnth ligase (New England Biolabs). The prepared adapter was then ligated to the cDNA pool using Thermostable 5´ App DNA/RNA Ligase (New England Biolabs) and again purified with the Qiagen MinElute kit. PCR amplification of cDNA library was carried out with Advantage PCR kit with primers that anneal to the 5 and 3′ adapter sequences. Samples were barcoded with overhanging reverse primers and amplified to PCR 18 cycles. DNA purification was carried out using AMPure beads (Beckman Coulter) and pooled at equimolar ratios. The PacBio SMRTbell prep kit was used for final library preparation according to the manufacturer (Pacific Biosciences) and sequenced on a PacBio Sequel II Long-Read Sequencer. Replicates 1 and 2 were sequenced on separate flow cells (1 flow cell each). For RNA seq: Extracted RNA was used to prepare RNA-seq libraries using the KAPA mRNA HyperPrep Kit (Kapa Biosystems). The RNA-seq library was sequenced on the Illumina Novaseq 6000 platform generating ~25 million paired-end reads per sample. For TimeLapse-seq: RNA was precipitated in isopropanol supplemented with 1 mM DTT and 20 μg glycogen and washed with freshly prepared 75% ethanol. Dried pellets were resuspended in nuclease-free water. Turbo DNase was used to deplete any remaining genomic DNA. RNAclean beads were used to isolate the RNA, which was then subjected to TimeLapse chemistry (2,2,2-trifluoroethylamine and mCPBA) and further purified with RNAclean beads. This recoded RNA was prepared for sequencing using the mammalian pico-input SMARTer stranded Total RNA-Seq kit v2 according to the manufacturer. The RNA-seq library was sequenced on the Illumina Novaseq 6000 platform generating ~30 million paired-end reads per sample.