GSM8775457: Tonsil, HPV-positive, sample5; Homo sapiens; RNA-Seq
Tonsil tissue collected from patient was finely minced by surgical scalpels. Minced tissue was moved to 50mL Conical tube and added with PBS up to 50mL volume.  After Centrifugation and removal of supernatant, Tissue pellet was added with 10mL of enzymatic cell dissociation buffer and incubated for 15 minutes in 37c incubator. [Cell Dissociation Buffer.  a-MEM 10mL + Collagenase type Ⅱ 20 mg (GIBCO) + Dnase 2 mg (Roche) +Dispase 40 mg(GIBCO)] Whole tissue-mixture was pipetted every 10 minutes to accelerate dissociation process. After passed through with 70um strainer, cell suspension was centrifuged at 300Xg for 10 minutes. 1mL of RBC Lysis Buffer(eBioscience) was treated to cell pellet and incubated for 1-5 minutes at Room Temperature. 50mL Conical tube which contains cell-buffer mixture was filled with PBS and centrifuged at 300x g for 10 minutes. Cells from pellet were re-suspended by MACS buffer (1X10 7 cells/80 uL) and mixed well for magnetic labeling with CD45 micro-beads (Miltenyi Biotec )(10 uL for 1X107 cells) at 4 °C for 15 minute. 10 mL of MACS BUFFER was added to cells and centrifuged. Cell pellet was re-suspended with MACS BUFFER (1X108 cells/ 500 uL) and applied onto the LS column (Miltenyi Biotec) placed in magnetic field of MACS Separator. [MACS Buffer. Steriled PBS contains 0.5% of FBS (v/v), 20mM EDTA] Unbound cell suspension was obtained for collecting CD45-negative population by magnetic separation. This magnetic separation step was conducted twice for increasing purity. Library was performed according to the manufacter's instructions (Chromium Next GEM single cell 3' v3.1 protocol, 10x genomics). Briefly, tonsil tissues 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 Illumina 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 appropriated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.