title: GSM8335815: PBMC_COVIDHC_CellPlex2_x8pool, GEX; Homo sapiens; RNA-Seq
library_construction_protocol: Cryopreserved PBMCs were thawed in a water bath at 37ºC and transferred to a 15 ml Falcon tube containing 10 ml of pre-warmed RPMI media supplemented with 10% FBS. Samples were centrifuged at 350 x g for 8 min at room temperature (RT), supernatant was removed and pellets resuspended with 1 ml of cold 1X PBS supplemented with 0.05% BSA. Samples were incubated during 10 min at RT with 0.1 mg/ml of DNAse I in order to eliminate ambient DNA and favor the resuspension of the pellet. Cells were filtered with a 40 µm strainer to remove eventual clumps and washed by adding 10 ml of cold PBS+0.05% BSA. Samples were centrifuged at 350 x g for 8 min at 4ºC and resuspended in an adequate volume of PBS+0.05% BSA in order to reach the desired concentration. PBMCs samples were multiplexed with 10X Genomics Cell Plex kit following the Cell Multiplexing Oligo Labeling for Single Cell RNA Sequencing Protocol (10x Genomics). Briefly, 0.2-1 million cells were centrifuged at 350x g at RT with a swinging-bucket rotor, resuspended in 100 µl of Cell Multiplexing Oligo and incubated at RT for 5 mins. Cells were washed 3 times with cold 1X PBS supplemented with 1% BSA, all centrifugations being performed at 350x g at 4C. Cells were finally resuspended in an appropriate volume of 1X PBS-1% BSA in order to obtain a final cell concentration. Multiplexed samples were encapsulated using the high throughput Chromium Next GEM Single Cell 3' HT Reagent Kit v3.1 in combination with the Chromium X instrument. Libraries were prepared following manufacturer's instructions of protocols CG000419.  Sequencing was carried out on a NovaSeq6000 system (Illumina) and NextSeq500 using the following sequencing conditions: 28 bp (Read 1) + 10 bp (i7 index) + 10 bp (i5 index) + 90 bp (Read 2), to obtain approximately 40.000 read pairs per cell for the Gene Expression (GEX) library and 2000-4000 read pairs per cell for the CellPlex library (CMO). scRNA-seq CellPlex 3' HT