GSM7011781: PBMC_H1-10X5; Homo sapiens; RNA-Seq
PBMC were isolation by using sodium heparin as an anti-coagulant. Mix thoroughly. Process within 24 hours, with best results obtained by prompt processing. If storing is needed before processing, store the blood at room temperature in the dark and on a rocker. Always keep cells at 20-37ºC. Pool the heparinized blood of individual donor and dilute blood 1:1 with Ca2+-free PBS (e.g., add 15ml PBS to 15ml blood). PBS should be at room temperature (20-37ºC). Gently mix by inverting the tubes/flasks two times. In a sterile, 50ml conical tube, add 15ml of Ficoll®. To avoid mixing the two phases, gently overlay the Ficoll® with 30ml of the diluted blood using a sterile serological pipette. Centrifuge the samples at 740g for 30 minutes at room temperature. After the centrifuge stops, immediately collect the mononuclear cells from the plasma/Ficoll® interphase and transfer into a sterile 50ml conical tube. Fill the tube to the 45ml mark with 37ºC CTL-Wash™ Medium. Centrifuge cells again, this time at 330g for ten minutes at room temperature. As soon as centrifugation is complete, cautiously decant the supernatant and discard it. Resuspend the cell pellet by tapping the tube until no clumps are visible. Pool cells of the same donor and count the cells (see Cell Counting above). The cell yield should be one-two million PBMC for each ml of blood drawn. Fill tubes to the 45ml mark with warm CTL-Wash™ (or CTL-Test™) Medium, and spin at 330g for ten minutes with the break on. RNA extraction was identified using TRIZOL RNA kits. Rinse cell monolayer with ice cold PBS once. Lyse cells directly in a culture dish by adding 1 ml of TRIZOL Reagent per 3.5 cm diameter dish and scraping with cell scraper. Pass the cell lysate several times through a pipette. Vortex thoroughly. The amount of TRIZOL reagent added is based on the area of the culture dish (1 ml per 10 cm2) and not on the number of cells present. An insufficient amount of TRIZOL Reagent may result in DNA contamination of the isolated RNA. Add 0.2 ml of chloroform per 1 ml of TRIZOL Reagent. Cap sample tubes securely. Vortex samples vigorously for 15 seconds and incubate them at room temperature for 2 to 3 minutes. Centrifuge the samples at no more than 12,000 x g for 15 minutes at 2 to 80C. Following centrifugation, the mixture separates into lower red, phenolchloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. Transfer upper aqueous phase carefully without disturbing the interphase into fresh tube. Measure the volume of the aqueous phase (The volume of the aqueous phase is about 60% of the volume of TRIZOL Reagent used for homogenization). Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of isopropyl alcohol per 1 ml of TRIZOL Reagent used for the initial homogenization.  Incubate samples at 15 to 30oC for 10 minutes and centrifuge at not more than 12,000 x g for 10 minutes at 2 to 4oC. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube. Remove the supernatant completely. Wash the RNA pellet once with 75% ethanol, adding at least 1 ml of 75% ethanol per 1 ml of TRIZOL Reagent used for the initial homogenization. Mix the samples by vortexing and centrifuge at no more than 7,500 x g for 5 minutes at 2 to 8 oC. Repeat above washing procedure once. Remove all leftover ethanol. Air-dry or vacuum dry RNA pellet for 5-10 minutes. Do not dry the RNA pellet by centrifuge under vacuum. It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility. Partially dissolved RNA samples have an A260/A280 ratio < 1.6. Dissolve RNA in DEPC-treated water by passing solution a few times through a pipette tip. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.