GSM5134522: Tbet Ho CD11b- rep2; Mus musculus; RNA-Seq
Spleen NK cells were purified by negative selection (Miltenyi MACS)  before flow cytometry-based sorting of NK1.1+ CD3-CD11b-CD27+ or NK1.1+  CD3-CD11b+CD27- cells, that were then processed for RNA extraction. RNA libraries were prepared as previously described (Picelli, S. et al. 2014). Briefly, total RNA was purified using the Direct-Zol RNA microprep kit (Zymo Research) according to manufacturer instructions and was quantified using QuantiFluor RNA system (Promega). 1µl of 10µM Oligo-dT primer and 1µl of 10mM dNTPs mix were added to 0.15ng of total RNA in a final volume of 2.3µl. Oligo-dT were hybridized 3min at 72°C and reverse transcription (11 cycles) was performed. PCR pre-amplification was then conducted using 16 cycles. cDNA were purified on AmpureXP beads (Beckman Coulter) and cDNA quality was checked on D5000 screen tape and analysed on Tape station 4200 (Agilent). 3ng of cDNA were tagmented using Nextera XT DNA sample preparation kit (Illumina). Tagged fragments were further amplified and purified on AmpureXP beads (Beckman Coulter). Tagged library quality was checked on D1000 screen tape and analyzed on Tape station 4200 (Agilent).