RNA-Seq of homo sapiens: human adult male memory B cells
Total RNA was isolated from cells of four HDs (age range 28-48 years) using RNeasy Plus Micro kit (Qiagen). The concentration of total RNA extracted was quantified using the Qubit RNA HS Assay Kit (ThermoFischer Scientific). The TruSeq RNA Sample Preparation v2 Kit (Illumina) was used for isolation of polyadenylated mRNA with oligo-dT beads, second strand cDNA synthesis and NGS library preparation. During the second strand synthesis, dUTP was incorporated in place of dTTP, thus preventing amplification of this strand during the subsequent PCR step and retaining strand information. Unique indexes, included in the standard TruSeq kit set A (Illumina) were incorporated at the adaptor ligation step for multiplex sample loading on the flow cells. The resulting constructs were purified by two consecutive AMPure XP beads cleanup steps and enriched by 15 cycles of PCR. Quality and quantity of the libraries were assessed using an Agilent DNA 1000 chip (Agilent Tecnologies) and Qubit dsDNA HS Assay Kit, respectively. Libraries were pooled in equimolar amounts (12 libraries/pool) and loaded onto High Output flow cell. The sequencing run was performed in paired-end mode (2 X 76-bp reads) using the Illumina NextSeq 500 platform. Base calling was performed by the instrument computer using Illumina Real Time Analysis (RTA) software that is integrated with NextSeq Control Software (NCS) and provides a summary of quality statistics as per Illuminas acceptance criteria for sequencing. CASAVA 1.8.2 was used for de-multiplexing and conversion of base calls to paired-end FASTQ files.