GSM6432760: CY3, Pantr2 Knockout, snRNA-seq; Mus musculus; RNA-Seq
Day E15.5 embryonic brains were collected from both wildtype and Pantr2-/- mice. The dorsal telencephalon was dissected, flash frozen in liquid nitrogen, and stored at –80°C until ready for nuclei isolation. Nuclei were isolated using a modified version of the Salty-EZ Lysis Buffer method outlined on protocols.io (https://www.protocols.io/view/protocol-for-nuclei-isolation-from-fresh-and-froze-kqdg36ybzg25/v5). In brief, tissues were lysed in 300μl of ice cold Salty-EZ10 lysis buffer containing 10mM Tris HCL pH 7.5, 146mM NaCl, 1mM CaCl2, 21mM MgCl2, 0.03% Tween-20, 10% EZ-lysis Buffer (Sigma Aldrich), 1mM DTT and 0.2U/μl Protector RNase Inhibitor. The tissue was gently triturated using a P200 wide-bore pipette tip on ice until the tissue was completely in solution. Once homogenized, 700μl of ice cold Slaty-EZ10 buffer was added and each sample was gently mixed using a wide-bore pipette tip. Next, the solution was spun down at 4°C at 500G for 5 minutes to pellet the nuclei in suspension. The supernatant was aspirated off and 500μl of WRB2 buffer containing 10mM Tris-HCl pH 7.5, 10mM NaCl, 3mM MgCl2, 1% w/v bovine serum albumin (BSA), 1mM DTT and 0.2U/μl Protector RNase Inhibitor. Nuclei were washed three times in 500μl WRB2 buffer before being used as the input material for 10X Genomics Chromium Single Cell V2 kit following standard protocol included with the kit. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, nuclei 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.