title: Illumina HiSeq 3000 paired end sequencing; CellMixS: quantifying and visualizing batch effects in single cell RNA-seq data
design: CellMixS: quantifying and visualizing batch effects in single cell RNA-seq data
library_construction_protocol: CR053, CR054, CR055, CR056, CR057, CR058: Fresh peripheral blood mononuclear cells (PBMCs) cells were harvested using Ficoll-Paque density gradient centrifugation from 15 ml whole blood specimens each of the two unrelated donors obtained from Blutspendezentrum SRK beider, Basel. Cells were washed with 1X PBS, resuspended in 20 ml 1X PBS and counted using trypan blue staining on Countess II (Life technology). For each donor PBMCs, three separate aliquots were prepared at 300 cells/ul suspended in 300 ul of RPMI-1640 plus 10%FBS. CR053/4 (fresh sample): Cells were centrifuged at 300xg for 10 minutes at room temperature, pellet  resuspended in 80ul PBS-0.04% BSA, and a total of 12,000 loaded on the 10x Single Cell B Chip. CR055/6 (MetOH fixation and cryopreservation):  4 volumes of MetOH pre-chilled at -20°C was added dropwise to cells and stirring with the micropipette tip. Cells were incubated for 30 minutes at -20°C and then stored at -80°C for 7 days.  CR057/8 (cryopreservation in 15% DMSO): Cells were centrifuged at 300xg for 10 minutes at room temperature, pellet resuspended in 500ul RPMI-1640 plus 40% FBS, then diluted using 500ul freezing medium (RPMI-1640 plus 40% FBS and 30% DMSO) pre-chilled at 4°C. The sample was placed inside a freezing container and stored at -80°C overnight before moving it to a liquid nitrogen tank for seven days storage.   After seven days of cryopreservation, samples were thawed and prepared for sequencing as described below. CR055/6 cells (fixed in MetOH) were first equilibrated at 4°C, followed by centrifugation at 3000xg for 10 minutes at 4°C, and the cell pellet finally resuspended in 50ul SSC cocktail (0.04% BSA, 1% RNAse inhibitor, 40mM DTT in SSC 1x). Cells were counted using trypan blue staining on Countess II (Life technology) and a total of 12,000 estimated cells were loaded on the 10x Single Cell B Chip. CR057/8 cells (stored in 15% DMSO) were thawed for few minutes at 37°C in a water bath, diluted 1:4 using RPMI-1640 plus 10% FBS, centrifuged at 300xg for 10 minutes at room temperature and the cell pellet resuspended using 50ul RPMI-1640 plus 10% FBS. Cells were counted using trypan blue staining on Countess II (Life technology) and a total of 12,000 estimated cells were loaded on the 10x Single Cell B Chip.  CR052, CR059, CR060 (CSF cells): 3ml of CSF each was collected from the diagnostic lumbar puncture of three patients at the University Hospital Basel. All three patients consented to CSF draw and the procedure performed according to Univ Hospital IRB guidelines.  Each CSF sample was centrifuged at 400xg for 10 minutes at the room temperature, the cell pellet was resuspended in 60ul RPMI-1640 plus 40% FBS, and cells were counted using trypan blue staining on Countess II (Life technology). Sample 1 (fresh CSF) freshly isolated cells were counted, assessed for cell viability, and finally loaded on the 10x Single Cell B Chip. Samples 2 and 3 (cryopreserved CSF) cells were each suspended in 450ul of RPMI-1640 plus 40% FBS, and then diluted using 500ul freezing medium (RPMI-1640 plus 40% FBS and 30% DMSO) pre-chilled at 4°C. Each sample was placed inside a freezing container and stored at -80°C overnight before moving it to a liquid nitrogen tank for 7 day storage. After seven days, the stored samples were thawed for few minutes at 37°C in a water bath, diluted 1:4 using RPMI-1640 plus 10% FBS and centrifuged at 300xg for 10 minutes at room temperature. The cell pellet was resuspended using 50ul RPMI-1640 plus 10% FBS. Cells were counted using trypan blue staining on Countess II (Life technology) and loaded on the 10x Single Cell B Chip if the concentration was higher than 200cells/ul and cell viability was above 60%.   CR038, CR039, CR040, CR041 (PBMCs): Fresh peripheral blood mononuclear cells (PBMCs) cells were isolated from whole blood specimens of four donors following similar protocol as described for CR053-CR058. In this data set, for each donor PBMCs, four separate aliquots were prepared at 500 cells/ul suspended in 200 ul of RPMI-1640 plus 10%FBS. CR038 (fresh sample) and CR039 (cryopreservation in 15%DMSO) cells were prepared as described for CR053-CR058.  CR040 (cryopreservation in CS10 media) and CR041  (cryopreservation in PSC media) cells were centrifuged at 300xg for 10 minutes at room temperature, cell pellet resuspended by dropwise addition and rocking the tube with 1ml pre-chilled CryoStor® CS10 medium (StemCell™ Technologies) for CR040 or 1 ml pre chilled PSC (ThermoFisher Scientific) for CR041. Cells were then placed inside a freezing container and stored at -80°C overnight before moving it to a liquid nitrogen tank for 7 day storage. CR039 cells thawing and library prep was done as described for CR053-CR058. CR040 (cryopreservation in CS10 media) cells were thawed for few minutes at 37°C in a water bath, diluted 1:4 using RPMI-1640 plus 10% FBS and centrifuged at 300xg for 10 minutes at room temperature. The cell pellet was resuspended in 50ul RPMI-1640 plus 10% FBS.  CR041 (cryopreservation in PSC media) cells were thawed for few minutes at 37°C in a water bath, diluted 1:4 using RPMI-1640 plus 10% FBS plus 0.01% RevitaCell (ThermoFisher Scientific) and centrifuged at 300xg for 10 minutes at room temperature. The cell pellet was resuspended in 50ul RPMI-1640 plus 10% FBS. For all samples, cells were counted using trypan blue staining on Countess II (Life technology) and a total of 12,000 estimated cells from each sample was loaded on the 10x Single Cell B Chip. cDNA libraries were prepared from all samples using the Chromium Single Cell 3' Library and Gel Bead kit v2/v3 (10x Genomics) according to the manufacturer's instructions. cDNA libraries were prepared from all samples using the Chromium Single Cell 3' Library and Gel Bead kit v2/v3 (10x Genomics) according to the manufacturer's instructions.