title: GSM8686491: CK3; Mus musculus; RNA-Seq
library_name: GSM8686491
library_construction_protocol: Cellular suspensions were loaded on a 10X Genomics GemCode Single-cell instrument that generates single-cell Gel Bead-In-EMlusion (GEMs). Libraries were generated and sequenced from the cDNAs with Chromium Next GEM Single Cell 3' Reagent Kits v3.1. Upon dissolution of the Gel Bead in a GEM, primers containing (i) an Illumina® R1 sequence (read 1 sequencing primer), (ii) a 16 nt 10x Barcode, (iii) a 10 nt Unique Molecular Identifier (UMI), and (iv) a poly-dT primer sequence were released and mixed with cell lysate and Master Mix. Barcoded, full-length cDNAs were then reverse-transcribed from poly-adenylated mRNA.                                                                             Total RNA was extracted using RNAsimple Total RNA Kit (DP419, TIANGEN). Silane magnetic beads were used to remove leftover biochemical reagents and primers from the post GEM reaction mixture. Full-length, barcoded cDNAs were then amplified by PCR to generate sufficient mass for library construction. R1 (read 1 primer sequence) were added to the molecules during GEM incubation. P5, P7, a sample index, and R2 (read 2 primer sequence) were added during library construction via End Repair, A tailing, Adaptor Ligation, and PCR. The final libraries contained the P5 and P7 primers used in Illumina bridge amplification.                                                           Libraries were constructed using U-mRNAseq Library Prep （AT4221,KAITAI-BIO） with Ribo-off rRNA Depletion Kit (Bacteria)(N407，Vazyme). Libraries were pooled and sequenced using the Illumina NovaSeq machine as 150-bp paired-end sequencing reads.