title: RNAseq of CpG-B stimulated B cells, Donor 2
design: Total RNA was extracted from frozen cell pellets using Qiagen RNeasy Plus Universal mini kit following manufacturers instructions (Qiagen, Hilden, Germany). Extracted RNA samples were quantified using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) and RNA integrity was checked with 4200 TapeStation (Agilent Technologies, Palo Alto, CA, USA). The RNA sequencing library was prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturers instructions (New England Biolabs, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were be fragmented for 15 minutes at 94 C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3ends, and universal adapters ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).
library_name: CpGB_Donor2