GSM8329361: gene expression - CELL_MIX_nc886; Homo sapiens; ncRNA-Seq
WPMY-1 and Hep3B cell lines were purchased from the American Type Culture Collection (Manassas, VA). To create Hep3B cells (designated "Hep3B-886") we used a lenticiral plasmd "pLL3.7.Puro.U6:nc886". This vector, a modification of the original lentiviral vector pLL3.7 (Addgene, Watertown, MA) was engineered to carry the nc886 gene (102 nts) under the control of the U6 promoter. Total RNA from these cell lines was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA). For the generation of a nc886 feature library, a gene-specific primer was designed to have 3' nc886 sequences, flanked by a feature barcode and a sequencing adaptor. The sequence is 5'-CGGAGATGTGTATAAGAGACAGNNNNNNNNNNGTATGTCCGCTCGATNNNNNNNNNAGGGTCAGTAAGCACCCGCG-3'. The first underlined 22 nts represent a Read2N adaptor (10X genomics); the second 15 nts, a feature barcode (TotalSeqTM-C0182, BioLegend); and the third 20 nts, complementary 3' nc886 sequences. The three nucleotide blocks are separated by 10 or 9 nts spacer sequences. Reverse transcription by this nc886-GSP will generate a 1st strand cDNA consisting of CCC-nc886-spacer-feature barcode-spacer-Read2N sequences. Second strand synthesis will be accomplished by the template switching oligos (TSOs) attached to the gel bead of 5' scRNA-seq reagent. Single cell suspensions of WPMY-1, Hep3B-886, and HEK293T cell lines were mixed in equal numbers and subjected to scRNA-Seq using Chromium Next GEM Single Cell V(D)J Reagent Kits v2 (10× Genomics, Pleasanton, CA). We set the cell recovery rate to 5000 per library and followed the manufacturer's instructions with a slight modification. During the GEM generation & barcoding step, we added 0.1 µM nc886-gene specific primer to the master mix. In addition to the 5' gene expression (GEX) library, nc886 feature library was constructed using the 5' Feature Barcode Kit (10× Genomics). Both libraries were sequenced on an Illumina Hiseq X as 100 bp paired-end.