GSM9009460: PBMCs, HFD + L-NAME 1; Mus musculus; RNA-Seq
Single cell preparation and barcoding: PBMCs were thawed according to 10x Genomics demonstrated protocol #CG00039 and a cell concentration of 700 to 1,200 PBMCs per µl of PBS + 0.04% BSA was set. Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1 (10x Genomics, #PN-1000128, Chip G: #1000127) was used to generate GEMs, reverse transcribe RNA and amplify cDNA according to manufacturer's instructions. Targeted cell recovery was 10,000. Library construction and sequencing: After cleanup (step 2.3), 2 µl of the cDNA was used for quality control on the Agilent fragment analyzer (DNF-473 Standard Sensitivity NGS Fragment Kit). In addition, 10 µl of the cDNA was used as input for step 3 of the Chromium Next GEM Single Cell 3´ Reagent Kit to perform fragmentation, end-repair, A-tailing and adapter-ligation. The finished library was monitored again using the fragment analyzer and the DNF-473 Standard Sensitivity NGS Fragment Kit. Equimolar amounts of each sample were plexed at 1.25 nmol/l. Sequencing was performed as paired-end 2x150bp (S4-Flowcell) on a Novaseq 6000 (Illumina).