title: GSM7673161: MU, brain cells, Mut, replicate 1, scRNAseq; Mustela putorius furo; RNA-Seq
library_construction_protocol: Somatosensory cortices were dissected carefully from fresh-frozen tissues stored in liquid nitrogen. Samples were minced into pieces <5 mm and then homogenized using a glass dounce tissue grinder (Sigma, D8938) in 2 mL of Nuclei EZ lysis buffer (Sigma, NUC-101) on ice. After two incubations on ice (with 4 mL of lysis buffer and 5 min each time), the homogenate was filtered through a 70 µm strainer, and then we used Debris Removal Solution (Miltenyi Biotech, 130-109-398) to perform density gradient centrifugation to clean the nuclear suspension according to the manufacturer's protocol. Isolated nuclei were resuspended and washed with nuclei suspension buffer (NSB, consisting of 1× PBS, 0.1% BSA and 0.4 U/µL Ambion™ RNase inhibitor (Thermo Fisher, AM2684)) and filtered through a 35 µm cell strainer. Nuclei were counted using a hemocytometer and diluted to 1000 nuclei/μL for optimal 10× loading. Approximately 8000 nuclei were targeted and captured for each reaction. All the libraries were prepared on a 10X GENOMICS platform following the RNA library preparation protocols. Cells were partitioned into nanoliter-scale Gel Bead-In EMulsions (GEMs) using 10x GemCode Technology, where a common 10x Barcode labeled all the cDNA produced from the same cell. Primers containing an Illumina R1 sequence (read1 sequencing primer), a 16-bp 10x Barcode, a 10-bp randomer, and a poly-dT primer sequence were released and mixed with cell lysate and Master Mix upon dissolution of the single cell 30 gel bead in a GEM. The GEMs were incubated, and barcoded, full-length cDNA was generated from poly-adenylated mRNA by reverse transcription. Then the GEMs were broken, and the leftover biochemical reagents and primers were removed with silane magnetic beads. Before constructing the library, the cDNA amplicon size was optimized by enzymatic fragmentation and size selection. After that, P5, P7, a sample index, and R2 (read 2 primer sequence) were added to each selected cDNA during end repair and adaptor ligation. P5 and P7 primers were used in Illumina bridge amplification of the cDNA (http://10xgenomics.com). Finally, 150-bp paired-end reads were sequenced from each library using the Illumina HiSeq4000. Concerning the update of the Single Cell 3' Regent Kits version, two libraries were prepared with Single Cell 3' Regent Kits V3.