FACS-sorted CD158b1b2jneg NK cells were lysed in 49 μL of RLT buffer (Qiagen) containing 1 μL of RNAse inhibitor (Thermo Fisher Scientific) and stored at -80°C.
The MicroRNAeasy KitTM with DNAse (Qiagen) was used to purify total RNA, which was then quantified by a Nanodrop 2000 (Thermo Fisher Scientific). Starting from 0.5 ng of high-quality total RNA with an RNA Integrity Number (RIN) greater than 6, assessed using a 4200 Tape Station (Agilent), libraries were prepared with the SMART-Seq Stranded Kit (Clontech-Takara). Libraries were then multiplexed in equimolar pools and sequenced by using a NextSeq-550 Illumina Platform. At least 60 million 75bp-paired-end reads per sample were generated. Read alignment, differential gene expression, and functional enrichment analyses were performed as previously described (16 (link)).
The MicroRNAeasy KitTM with DNAse (Qiagen) was used to purify total RNA, which was then quantified by a Nanodrop 2000 (Thermo Fisher Scientific). Starting from 0.5 ng of high-quality total RNA with an RNA Integrity Number (RIN) greater than 6, assessed using a 4200 Tape Station (Agilent), libraries were prepared with the SMART-Seq Stranded Kit (Clontech-Takara). Libraries were then multiplexed in equimolar pools and sequenced by using a NextSeq-550 Illumina Platform. At least 60 million 75bp-paired-end reads per sample were generated. Read alignment, differential gene expression, and functional enrichment analyses were performed as previously described (16 (link)).
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