title: GSM6047633: P3-NI-scRNAseq; Homo sapiens; RNA-Seq
library_construction_protocol: The resected tumor samples were washed by phosphate buffered saline (PBS) and completely immersed in MASC tissue storage solution (Miltenyi, Germany) at 4 ℃.Tumor dissociation for 10X Single Cell RNA Sequencing according to protocol (CG000147, https://assets.ctfassets.net/an68im79xiti/2s0LZ3yI8OMEheRtFo9Szc/1fbfbd97ec3e574b6ab8b8fa12eef0cd/CG000147_Demonstrated_Protocol_Tumor_Dissociation_RevB.pdf). Using a wide-bore pipette tip, gently and thoroughly mix the cell suspensions. Determine the cell concentration using a Countess® II Automated Cell Counter. Centrifuge cells at 300 rcf for 5 min. Remove supernatant without disrupting the cell pellet. Using a wide-bore pipette tip, add 1 ml 1X PBS with 0.04% BSA to the tube. Gently pipette mix 5 times and invert tubes to resuspend the cell pellet. 10x Genomics (US) Single Cell Protocols (CG000053). Cell Counting & Quality Control. Thoroughly vortex 0.4% trypan blue stain and centrifuge briefly. Using a wide-bore pipette tip, gently and thoroughly mix the cells. Immediately, aliquot 10 µl cells and mix with 10 µl 0.4% trypan blue stain. Gently pipette mix. Transfer 10 µl trypan blue stained cells to a chamber on Countess® II Cell Counting Slide and determine the cell concentration and cell viability. The single cell suspension was loaded into Chromium microfluidic chips with 3' v2 chemistry and barcoded with a 10× Chromium Controller (10X Genomics, US). RNA from the barcoded cells was subsequently reverse-transcribed and sequencing libraries constructed with reagents from a Chromium Single Cell 3' v2 reagent kit (10X Genomics, US) according to the manufacturer's instructions (CG000108). The Chromium™ Single Cell 3' v2 Protocol (CG00052) produces Single Cell 3' libraries, ready for Illumina Novaseq PE150 platform. 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.