title: GSM5237042: Sample_04_healthy; Homo sapiens; RNA-Seq
library_construction_protocol: Tissue was collected in 5ml of RPMI media. Further disaggregation of tissue into a single-cell solution for sequencing was completed using the MACs tumor dissociation kit (Miltenyi Biotec) with the standard tough tumor protocol. Brieﬂy, the MACs tumor dissociation kit enzyme mix (300μl) was added to each sample. Next, samples were put into the gentleMACs Dissociator and ran through the tough tumor program. The cell suspension was then applied to a 70um cell strainer. Cells were pelleted, counted and frozen according to the slow freezing procedure in standard cryo-vials (1 ml cell suspension in the cryopreservation medium). Next, the cells were thawed, washed in PBS, stained with Live/dead Aqua Fluorescent Reactive Dye (Life Technologies) and anti-mouse CD45 antibody (30-F11, BD Biosciences) at 4°C for 20 minutes and sorted on a FACS Aria III (BD Biosciences) with a 100µm nozzle. Purity of live CD45+/CD45- cells was assessed after sorting, prior to downstream use. CD45+/CD45- cells were resuspended in 1ml PBS plus 0.04% BSA and washed two times by centrifugation at 450 rcf for 7min. After the second wash cells were resuspended in 30 ul and counted with an automatic cell counter (Countess II, Thermo Fisher) to get a precise estimation of total number of cells recovered.   Afterwards CD45+/CD45- cells of each sample were loaded into one channel of the Single Cell Chip A using the Single Cell 3' v2 single cell reagent kit (10X Genomics) for Gel bead Emulsion generation into the Chromium system. Following capture and lysis, cDNA was synthesized and amplified for 14 cycles following the manufacturer's protocol (10X Genomics). 50 ng of the amplified cDNA were then used for each sample to construct Illumina sequencing libraries. Sequencing was performed on the NextSeq500 Illumina sequencing platform following 10x Genomics instruction for reads generation.