GSM6693249: Mw_Cs3; Homo sapiens; RNA-Seq
Peripheral blood mononuclear cells (PBMC) were isolated from whole blood samples by density gradient centrifugations using HiSep™ LSM 1077 media (Himedia, Mumbai, India). 5 x 106 PBMCs were used for RNA isolation using the Trizol method and followed by DNase treatment for RNA purification. Purified RNA was quantified using a Qubit 4.0 fluorometer. Take 5μg of total RNA used for the polyA RNA selection using NEB NEXT oligo d(T)25 beads (NEB, MA, USA). PolyA RNA was quantified and integrity assessed using Qubit 4.0 fluorometer (Invitrogen, Waltham, Massachusetts, United States) according to manufacturer instructions. Libraries were prepared according to Oxford nanopore technologies protocol accompanying the Direct cDNA Native Barcoding Sequencing kit (SQK-DCS109 with EXP-NBD104, Oxford, UK). 100ng of polyA RNA was used for the library preparation. Using H minus Reverse Transcriptase (Invitrogen, Waltham, Massachusetts, United States), complementary DNA (cDNA) was synthesised, followed by RNA degradation and second-strand synthesis of cDNA. Double-stranded cDNA was used for the end preparation, followed by native barcoding and adaptor ligation (all steps were followed, according to the manufacturer's instructions). Ligated cDNA was loaded on the flow cell (R9.4.1) in MinION and were sequenced specifying 72 hours protocol. MinKNOW (v21.06.10, Microsoft Windows OS based) was used to generate FAST5 files.