GSM7081457: HN02_R; Homo sapiens; RNA-Seq
Isolated monocytes and neutrophils were kept in RLT buffer (Qiagen) at -80°C till RNA isolation. Total RNA was isolated using the RNeasy@ Mini kit (Qiagen) as per the manufacturer's instructions . The purity of RNA was assessed in-house by a Nanodrop (Thermo Scientific) spectrophotometric measurement. The samples with a 260/280 ratio between 1.9 and 2.0 were processed for RNA sequencing. A NEB Ultra II Directional RNA-Seq Library Prep kit protocol was used to prepare libraries for total RNA sequencing (NEB, Cat# E7760L). An initial Concentration of 200ng of total RNA was taken for the assay. First, the ribosomal RNA (rRNA) which constitutes ~95% of the total RNA population was removed using biotinylated, target-specific oligos combined with Ribo-Cop rRNA removal beads. Following purification, the ribodepleted RNA was fragmented using divalent cations under elevated temperatures. The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase. Second strand cDNA synthesis was performed using DNA Polymerase I and RNase H enzyme. The cDNA fragments were then subjected to a series of enzymatic steps that repair the ends, tails the 3' end with a single 'A' base, followed by ligation of the adapters. The adapter-ligated products were then purified and enriched using the following thermal conditions: initial denaturation of 98°C for 30sec; 13 cycles of - 98°C for 10sec, 65°C for 75sec; final extension of 65°C for 5mins. PCR products were then purified and checked for fragment size distribution on Fragment Analyzer using HS NGS Fragment Kit (1- 6000bp) (Agilent, Cat# DNF-474-1000) and a few samples were checked for fragment size distribution on TapeStation using D1000 DNA ScreenTapes (Agilent, Cat# 5067-5582). The libraries for all the samples were prepared with paired-end chemistry and a length of 150 bp. Quantification of all libraries was carried out using Qubit High Sensitivity Assay (Invitrogen, Cat# Q32854). The obtained libraries were pooled and diluted to the final optimal loading concentration before cluster amplification on the Illumina flow cell. Once the cluster generation was completed, the clustered flow cell was loaded on the Illumina HiSeqX instrument to generate a minimum of 100M raw reads per sample