GSM9822467: Hehd1_CHR_Rep2; Homo sapiens; RNA-Seq
700,000 hiPSCs or hESCs were used for RNA extraction for each biological replicate. Cells were washed once with 1ml Dulbecco's Phosphate Buffered Saline (DPBS) and harvested using 500µl Accutase (StemCell Technologies, 07922) for 3 minutes at 37°C. Dissociated cells were then collected with 5ml of corresponding hPSC medium and centrifuged for 3 min at 400 RCF at RT. Supernatant was aspirated and the cells were resuspended in 1ml cold DPBS. RNA extraction from hiPSCs/hESCs was conducted using the Monarch Total RNA Miniprep Kit (NEB T2010S) according to the manufacturer's protocol, with DNase I treatment used to enzymatically remove residual gDNA. RNA concentration was quantified using Nanodrop and the RNA Integrity Number (RIN) was assessed using the Agilent RNA 6000 Pico Kit (Agilent, 5067-1513) on Agilent 2100 Bioanalyzer (Agilent). A RIN value of >7 was deemed to be of good quality and used for downstream RNA-seq library processing. The isolated RNA was frozen and stored at -80°C until RNA-seq library preparation. RNA-seq library preparation was performed on the isolated RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module, which isolated mRNA from total RNA (100ng input) by poly(A)-enrichment. RNA-seq libraries were subsequently prepared from the isolated mRNA using the NEBNext Ultra II Directional RNA Library Prep Kit from Illumina, the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490L), and the NEBNext Multiplex Oligos from Illumina (Index Primers Set 1, 2, 3 and 4, NEB, E7335S, E7500S, E7710S, E7730S), following the manufacturer's protocols. The final sequencing-ready libraries were amplified using the protocol's PCR programme with 12 cycles. Size distribution and yield quantification of the RNA-seq libraries were assessed using an Agilent 2100 Bioanalyzer high sensitivity DNA chip (Agilent 5067–4626).