Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Klf12-/- Lymphocyte Transcriptomes

The goal of this study is to determine the role of the transcription factor, Klf12, in mouse NK cells. Transcriptome profiling by RNA-Seq of wild-type and Klf12-deficient mouse CD4+ T cells, CD8+ T cells, B cells, and NK cells. We found that Klf12-deficient mouse NK cells express more transcripts of Btg3 and have a proliferative impairment compared to wild-type mouse NK cells in a competitive setting. Total RNA from 6 week-old Klf12+/+ and Klf12-flox/flox beta-actin Cre- littermate males was isolated from splenic CD3-NK1.1+ NK cells, CD4+TCRβ+CD25- T cells, CD8+TCRβ+CD25- T cells, and B220+IgM+IgD+ B cells following the ImmGen standard protocol (https://www.immgen.org/). One thousand cells (≥ 99% purity) were double-sorted directly into 96-well plates containing TCL buffer (Qiagen) and 1% beta-mercaptoethanol, frozen, and analyzed by the Broad Technology Labs for SmartSeq2 library preparation and NextSeq500 sequencing. Transcripts were quantified using Cuffquant and then normalized using DEseq (Immological Genome Consortium). Three male mice were used per genotype.