GSM8148786: KS_II_age_control; Homo sapiens; RNA-Seq
Venous blood samples of 4mL were collected in Vacutainer ACD tubes (BD Biosciences) and transferred into 15mL Falcon tubes (Thermo Fisher Scientific). Samples were diluted with 4mL of serum-free wash buffer (PBS (-Ca2+/-Mg2+). 4.5mL of Ficoll Histopaque was added to 15mL SepMate Tubes (Stemcell Technologies). 8mL of diluted blood was gently layered on top of the Ficol Histopaque using auto pipette, ensuring separation of layers. The samples were centrifuged at 1200 x g at room temperature for 15 min. ~1mL of PBMC was aspirated from the interphase between histopaque and the medium and into a 50mL Falcon tube. The remaining supernatant was decanted into the Falcon tube and PBS buffer was added up to 50mL. Samples were washed and centrifuged twice at 250 x g at room temperature for 6 min. The cells were counted. The supernatant was removed without disturbing the pellet, and the pellet was resuspended in 200uL 1X Lysis Buffer with Detergent + 2uL phenylmethylsulfonyl fluoride in ethanol (PMSF). Samples were vortexed for 20 sec and heated at 85°C for 10min in heating block. Samples were transferred to -80°C freezer. Culture media was prepared using 44mL RPMI (1640 Medium – Thermo Fisher Scientific) and 5mL (10%) fetal bovine serum (FBS – Thermo Fisher Scientific) and 500uL GlutaMAX (Thermo Fisher Scientific). Wash buffer was prepared using Dulbecco's phosphate-buffered saline (DPBS) and 2% FBS. 10mL of culture media was added to a 15mL Falcon tube. PBMC aliquots were thawed in 37°C water bath and transferred to the Falcon tube with culture media. Samples were centrifuged at 250 x g at room temperature for 10 min and the supernatant was aspirated without disturbing the pellet. Samples were resuspended in 3mL of wash buffer. A 15uL counting aliquot was taken and 10mL of wash buffer was added before centrifugation at 250 x g at room temperature for 10 min. Cells were counted in the sample (using manual hemocytometer. The supernatant was aspirated and cells resuspended in wash buffer such that final concentration was 1 million cells/uL. PBMCs were sorted into a single cell suspension of live cells using the BD FACSAria™ III Cell Sorter and loaded onto a Chromium Next GEM Chip G (10X Genomics) for a target output of 10,000 cells per sample. Single-cell droplet capture was performed on the Chromium Controller (10X Genomics) and the cDNA library was prepared in accordance with the Chromium Next GEM Single Cell 3' v3.1 (dual index) protocol The samples were sequenced by AZENTA Life Sciences (China) on an Illumina HiSeq4000 instrument (2x150 base pair paired end run) for a depth of 20,000 reads per cell