GSM5579581: SNORD126_ASO1_30_n1; Homo sapiens; RNA-Seq
Total RNA extraction from transfected HepG2 cells was performed using RNeasy mini kit (Qiagen) as recommended by the manufacturer including on column DNase digestion with RNase-Free DNase Set (Qiagen). However, 1.5 volumes Ethanol 100% was used instead of the recommended 1 volume ethanol 70% in order to retain smaller RNA. RNA integrity of each sample was assessed with an Agilent 2100 Bioanalyzer. RNA was reversed transcribed using Transcriptor reverse transcriptase (Roche) and knockdown levels were evaluated by qPCR. RNAseq librairies were generated from 1ug DNA-free total RNA/condition using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (E7760S) and following the Protocol for use with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) . The resulting ibrairies were submitted to a total of 10 cycles of amplification then purified using 0.9X Ampure XP beads. Quality and size was assessed with an Agilent 2100 Bioanalyser. Librairies were then quantified using a Qubit fluorometer, pooled at equimolar concentration and 1.8pM was sequenced on Illumina's NextSeq 500 using a NextSeq 500/550 High Output Kit v2.5 (150 cycles) paired-end 2x75bp.