title: GSM8630805: FNA20; Homo sapiens; RNA-Seq
library_construction_protocol: The aspirate samples volumes are usually from ~0.1 to 0.2 ml and often contain substantial amounts of blood. The physician on-call obtained the samples, and washed one of the aspirates directly into a 6 ml heparinized blood draw tube containing 5 ml of ice cold complete RPMI cell culture medium. Cells were pelleted by centrifugation (5,000 rpm for 5 min.).  The supernatant was removed into bleach, and the pellet was re-suspended in 1 X RBC NH4Cl lysis buffer on ice and occasionally mixed gently for 3 min. Following centrifugation, the sample was resuspended in 1 ml of Accutase™ solution (Sigma-Aldrich) and incubated with occasional gentle mixing for another 2 min. on ice. Following centrifugation, the final cell pellet was gently re-suspended to a single cell suspension in 0.2 ml ice cold saline.  To this cell suspension, 1 ml of ice cold 100% methanol was added dropwise with constant mixing to fix and sterilize the sample for transport. A whole sample or aliquot of the MeOH fixed sample was centrifuged (600xg) the supernatant was discarded in medical waste, and the sample resuspended in PBS with 1% BSA, the sample was filtered through 70 and 40 micron FlowMI filters and submitted for 10X genomics sequencing. Libraries were prepped according to the 10X Genomics scRNA-seq 3' v3.1 Next GEM protocol, NovaSeq Reagent Kit v1.5_150x150 bp Sequencing.  All TB and HIV genes are listed with TB-'gene_name' and HIV1-'gene_name', respectively, in the counts matrix.