GSM2631812: mu_CD_IL6Raflox_Liver replicate 3; Mus musculus; RNA-Seq
NK cell subpopulations from murine organs and blood or human blood were purified from single cell suspensions using FACSAria-III or FACSAria-Fusion cell sorters (BD Bioscience) after immunostainings as described above. In mice, NK cells were sorted from obese versus lean animals identified as single, viable CD45+ CD3- NK1.1+ CD11b+ mature NK cells. Final sorting gates of human NK cells identified IL6Ra+ versus IL6Ra- CD3- CD56+ single cells. Purified cells were directly sorted into RNA-protect cell reagent (Qiagen, Germany).   Stabilized, purified NK cell populations were pelleted by centrifugation and total RNA was extracted using the Arcturus RNA picopure Kit (KIT0204, ThermoFisher Scientific) following the manufacturer´s instructions. RNA integrity was assessed with the Agilent 2100 Bioanalyzer. For organs and bulk sorted NK cells: RNA libraries were prepared from a minimum of 100ng total RNA using the TruSeq® RNA sample preparation Kit v2 (Illumina). Complementary DNA (cDNA) was transcribed from poly-A selected RNA, which served for library generation. Libraries were sequenced in replicates for 30 million reads on an Illumina HiSeq 2000 sequencer with a paired-end (101x7x101 cycles) protocol. Single cells were sorted in 384-well plates containing 240nL of primer mix and 1.2uL of PCR encapsulation barrier, Vapor-Lock (Qiagen GmbH, Germany). Single cell RNA sequencing was performed using CEL-Seq2 method (Hashimshony et al., 2016) with several modifications.  Importantly, a fivefold volume reduction was achieved using a nanoliter-scale pipetting robot, Mosquito HTS (TTP Labtech).  Sorted plates were centrifuged at 2200g for 10min at 4°C, snap-froze in liquid nitrogen and stored at -80°C until processed. 160nL of reverse transcription reaction mix and 2.2μl of second strand reaction mix was used to convert RNA into cDNA. cDNA from 96-cells was pooled together before clean up and in vitro transcription, generating 4 libraries from one 384-well plate. 0.8μl of AMPure/RNAClean XP beads (Beckman Coulter GmbH, Germany) per 1μl of sample were used during all the purification steps including library cleanup. 8 libraries (768 single cells) were sequenced in a single lane (pair-end multiplexing run, Read 1 30bp Read 2 120bp read length) of Illumina NextSeq 500 sequencing system generating 200 million sequence fragments.
GEO Accession
GSM2631812