title: GSM8393392: day 13 ATIIs from young and old mice_hto; Mus musculus; RNA-Seq
library_name: GSM8393392
library_construction_protocol: RNA was extracted using RNeasy kits (Qiagen, 74104). RNA quantity and quality was measured using a Nanodrop (Thermo Scientific). 100ng RNA per sample was used to make cDNA using the iScript™ cDNA Synthesis Kit (BioRad, 1708890) We sorted Sytox blue negative, live cells for scRNA-seq using the Chromium Next GEM Single Cell 3ʹ Reagent Kit v3.1 (Dual Index, 10x Genomics, 1000269) with Feature Barcode technology for Cell Multiplexing (10x Genomics, 1000262). Libraries were prepared according to the manufacturer's instructions. Prior to loading cells on the microfluidic chip, we performed quality control and cell counting. All cell suspensions were >75% viable and within the concentration range recommended by the manufacturer. Following GEM generation and barcoding via reverse transcription (RT), we performed post GEM-RT cleanup and cDNA amplification. Subsequently we proceeded with gene expression and cell multiplexing library construction, performed QC on the generated libraries and submitted them for sequencing. Gene expression libraries were pooled with cell multiplexing libraries and sequenced on the NovaSeq 6000 (Illumina, San Diego, CA) to generate a minimum 2,000 reads per cell for cell multiplexing libraries and 20,000 reads per cell for gene expression libraries in a 28/10/10/90 (R1/i7/i5/R2) bp configuration according to 10X Genomics guidelines.