GSM6254974: LR UM Case14; Homo sapiens; RNA-Seq
For short reads sequencing, total RNA was isolated using Trizol Reagent (Invitrogen Life Technologies), then the concentration, quality and integrity of RNA were determined by NanoDrop spectrophotometer (Thermo Scientific). Three micrograms of RNA were used as input material for the RNA sample preparations. For long reads sequencing, total RNA was isolated using the Trizol Reagent (Invitrogen Life Technologies), and the concentration, quality and integrity were determined by NanoDrop spectrophotometer (Thermo Scientific). For short reads sequencing, sequencing libraries were generated according to the following steps. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. First strand cDNA was synthesized using random oligonucleotides and Super Script II. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed. After adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments of the preferred 400-500 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in PCR reaction. Products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). For long reads sequencing, A total of 1ug RNA was prepared for cDNA libraries using cDNA-PCR Sequencing Kit (SQK-PCS109) according to the instructions of Nanopore Technologies (ONT). Defined PCR adaptors were directly added to both ends of the first-strand cDNA by reverse transcriptase. After 14-cycle of PCR by LongAmp Tag (Martin,  #21), the PCR products were subjected to ONT adaptor ligation using T4 DNA ligase (Martin,  #21). Agencourt XP beads was used for DNA purification according to ONT protocol. The final cDNA libraries were added to FLO-MIN109 flow-cells and were sequenced on PromethION platform.