GSM7765988: CD45+ cells of paracaspase-deficient Malt1 overexpression tumor; Mus musculus; RNA-Seq
Single cell suspensions isolated from tumors developed from E0771-Vector control tumor tissue, E0771 Malt1-WT overexpression tumor tissue and E0771 paracaspase-deficient Malt1 (Malt1-PD) overexpression tumor tissue. About 10000 CD45+ live cells were sorted from tumors of each strain by BD FACS Aria ii. Reverse transcription, cDNA amplification and library preparation were performed using the Chromium Single Cell V(D)J Reagent Kits (10x Genomics, includes Cat# PN-1000006, Cat# PN-1000020, Cat# PN-1000009, and Cat # PN-120262) according to manufacturer's protocol. Pair-end 150 bp sequencing was performed on Illumina HiSeq X Ten platform by Novogene Co.,Ltd. Single cell sequence data was pre-processed with Cell Ranger 3.0.2 (10x Genomics). Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, CD45+ cells in each group 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.