GSM5462410: RNA2InjCD27pos CD11bneg; Mus musculus; RNA-Seq
BM cells from five mice per group (control or FLB1 injected) were pooled per experiment prior to NK cell sorting, and three independent experiments were performed. NK cells were first sorted using EasySep™ Mouse NK Cell Isolation Kit (STEMCELL Technologies), then with Aria II on the basis of live/CD3-CD19-NK1.1+CD27+CD11b± gating. RNA was isolated from sorted cells using the RNeasy micro plus kit (Qiagen) and mRNA quality was evaluated using an Agilent 2100 (Pico Chip). Full length cDNA were generated from 4ng of total RNA using Clontech SMART-Seq v4 Ultra Low Input RNA kit for Sequencing (Takara Bio Europe, Saint Germain en Laye, France) according to manufacturer's instructions with 10 cycles of PCR for cDNA amplification by Seq-Amp polymerase. Six hundreds pg of pre-amplified cDNA were then used as input for Tn5 transposon tagmentation by the Nextera XT DNA Library Preparation Kit (96 samples) (Illumina, San Diego, CA) followed by 12 cycles of library amplification. Following purification with Agencourt AMPure XP beads (Beckman-Coulter, Villepinte, France), the size and concentration of libraries were assessed by capillary electrophoreris. The libraries were sequenced on Illumina Hiseq 4000 sequencer as Single-Read 50 base reads following Illumina's instructions. Image analysis and base calling were performed using RTA 2.7.3 and bcl2fastq 2.17.1.14. Adapter dimer reads were removed using DimerRemover