GSM5342451: ZT 0 KO IgA+ PC replicate 1; Mus musculus; RNA-Seq
Collagenase D and Dnase I isolation of single cell small intestinal lamina propria cells (see methods of above manuscript) Sort-purfication of CD138+ IgA+ Plasma Cells via fluorscence activated cell sorting (FACS) Samples lysed in Qiagen RLT buffer and processed as per manufacturer's instructions (RNA mini kit) Normalised RNA was used to generate libraries using NEB Next Ultra RNA library Prep Kit (Illumina). Indices were included to multiplex samples and mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesised using random hexamer primers followed by second strand cDNA synthesis. Following end repair, A-tailing, adaptor ligation and size section libraries were further amplified and purified and insert size validated on an Agilent 2100, and quantified using quantitative PCR (qPCR). Libraries were then sequenced on an Illumina NovaSeq 6000 S4 flowcell with PE150 according to results from library quality control and expected data volume.
GEO
GSM5342451