GSM7784875: Mature3; Tupaia belangeri; RNA-Seq
The retina was carefully dissected out from TS with strict compliance to the ethical guidelines. In detail, the dissected tissues were washed with cold phosphate buffered saline (PBS; Invitrogen, Carlsbad, USA), quickly frozen and then stored in liquid nitrogen before use. Preceding the library construction process, the tissues were first thawed, cut into small pieces, and then transferred to 1.5 mL tube contained with 1× homogenization buffer containing 30 mmol/L CaCl2, 18 mmol/L Mg(Ac)2, 60 mmol/L Tris-HCl (pH 7.8), 320 mmol/L sucrose, 0.1% nonidet P-40, and 0.1 mmol/L ethylene diamine tetraacetic acid (Invitrogen). The tissue pieces werethen transferred to a 2 mL Dounce homogenizer and stroke on the ice with loose pestles and then with tight pestles for 15 times. The nucleus extraction was filtered with 40 mm strainer and spanned down at the speed of 500 g for 10 min at 4 ℃ to carefully discard the supernatant. The pellets were resuspended with PBS containing 0.1% bovine serum albumin (Invitrogen) and 20 U/mL RNase Inhibitor for later 10x Genomics 30 library construction (10x Genomics, Pleasanton, USA). Library was performed according to the manufacter'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 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.