title: GSM7114954: Blood, gene expression_1; Homo sapiens; RNA-Seq
library_construction_protocol: Tumor samples were transported in RPMI stored at 4°C, rinsed with PBS, minced and incubated in a rotation shaker for 40 minutes at 37°C in Collagenase IV 0.25mg/ml, Collagenase D 200U/ml and DNAse I 0.1mg.ml (all Sigma). Cell suspensions were then aspirated through a 18G needle ten times and strained through a 70-micron mesh prior to RBC lysis. Dead cells were removed using an EasySep Dead Cell Removal (Annexin V) Kit. Cell suspensions were sorted into CD45+ and CD45- cells using the EasySep™ Human CD45 Depletion Kit per kit instructions. PBMCs from the blood samples were isolated within 3 hours of collection via Ficoll density gradient centrifugation for 10 min at 1200g room temperature. The supernatant was then spun down at 500g for 10 min at 4 degrees C and the pellet resuspended to a concentration of 10x10^6 cells/ml cold Human Serum AB (GemCell HAB and HAB + 20% DMSO in 1:1 ratio). The resulting PBMCs were stored in 2ml Cryogenic vials in liquid nitrogen. Previously frozen PBMCs were thawed, washed 2X in RPMI 2% FCS, treated with ACK lysis buffer (Lonza) to remove RBCs and briefly incubated with DAPI. 300,000 cells were then sorted on a DAPI negative gate. Cells were then stained for 30 minutes at room temperature with a panel of 138 Total-Seq-C antibodies (Biolegend, Stoeckius et al 2017) and washed 3x using the HT1000 laminar wash system (Curiox). Cells were then counted using the Cellaca MX High-throughput Automated Cell Counter as described in the manufacturer's protocol (Nexcelom), pooled and loaded on the 10x Chromium Next GEM Chip K Kit using a superloading strategy mixing cells from the same sample across lanes. For tumor samples, 10x libraries were constructed using the 10x supplied protocol and sequenced at the Mount Sinai Genomics Core Facility. For PBMCs in the blood samples, libraries were prepared according to manufacturer's protocol (10x Genomics). scRNA-seq, CITE-seq (blood only), TCR-seq