GSM7062618: Sample 10, female biol rep 6, snRNAseq; Mus musculus; RNA-Seq
Mechanical -Detergent Lysis Protocol described by Matson et. al. 2018 (JoVe) was used to create nuclei suspensions. Fresh PVT tissue punches were collected and mechanically lysed in lysis buffer using a Dounce homogenizer tube. Crude nuclei preparation were filtered and washed using low sucrose buffer. Nuclei prep were then centrifuged and reconstituted using low sucrose buffer. Following mechanical homogenization, nuclei prep were pased through a sucrose density gradient to cleanse the sample of debris. Following centrifugation, the nuclei were resuspended in a 0.04%BSA solution for sequencing. Library was performed according to the manufacter's instructions (single cell 3' v3 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 the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.