GSM9633389: Brain Sample_male 2; Mus musculus; RNA-Seq
Single nucleus were suspended in calcium-, magnesium- and RNase-free PBS containing 0.04% weight/volume BSA. Then Cell nucleus were added to each channel. After generation of GEMs, reverse transcription reactions were engaged barcoded full-length cDNA followed by the disruption of emulsions using the recovery agent and cDNA clean up with DynaBeads Myone Silane Beads. Subsequently, the amplified cDNA was fragmented, end-repaired, A-tailed, and index adaptor ligated and library amplification.RNA libraries were prepared with Chromium Single cell 3' Reagent v3 Kits according to the manufacturer's protocol. 10x Genomics Chromium Single Cell 3' Reagents v3 snRNAseq Library preparation was performed according to the manufacturer's instructions (Single Cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate gel bead-in-emulsions (GEMs). Each GEM contained a single cell, a gel bead, and the necessary reagents for the reaction. The poly-A RNA released from the cell lysate within each GEM bound to primers on the gel bead and was reverse-transcribed into cDNA, which contained an Illumina R1 sequence, a Unique Molecular Identifier (UMI), and a 10x Barcode. Subsequently, the pooled barcoded cDNA was cleaned up with Silane DynaBeads, amplified by PCR, and appropriate-sized fragments were selected with SPRIselect reagent for subsequent library construction. During library construction, an Illumina R2 primer sequence, P5/P7 adapter sequences, and a sample index were added to generate a complete library ready for sequencing. The final library was assessed for concentration and fragment size distribution, and the qualified library was sequenced on the Illumina NovaSeq high-throughput sequencing platform.