GSM9023071: IgG2_Donor 1; Homo sapiens; RNA-Seq
FACS-separated B cell subsets from 8 donors were sorted into DNA LoBind tubes (Eppendorf) and washed with PBS. Total RNA was isolated from B cell sorted subsets using Qiagen RNeasy Plus Universal mini kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). RNA samples were quantified using Qubit 4.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked with RNA Kit on Agilent 5300 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA). Total RNA samples that had inadequate quantity or an RNA Integrity Number (RIN) < 8 were excluded from the library preparation. RNA sequencing library preparation was prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina following manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented. First strand and second strand cDNA were subsequently synthesized. The second strand of cDNA was marked by incorporating dUTP during the synthesis. cDNA fragments were adenylated at 3'ends, and indexed adapter was ligated to cDNA fragments. Limited cycle PCR was used for library amplification. The dUTP incorporated into the cDNA of the second strand enabled its specific degradation to maintain strand specificity. Sequencing libraries were validated using NGS Kit on the Agilent 5300 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA). The sequencing libraries were multiplexed and loaded on the flowcell on the Illumina NovaSeq 6000 instrument according to the manufacturer's instructions. The samples were sequenced using a 2x150 Pair-End (PE) configuration v1.5.