GSM9127972: Control 03, Kidney; Homo sapiens; RNA-Seq
For single nucRNA-sequencing, snap frozen biopsies were thawed and chopped using a fresh razor blade. After adding 200 µl lysis buffer (1 mM DTT, 0.1% Triton X-100, 0.2 U/µl SUPERaseIN RNAse Inhibitor and 0.4 U/µl RNasin® Plus Ribonuclease Inhibitor in Nuclei PURE Lysis Buffer), the tissue homogenized 60 times using a pestle on ice. Next, the lysate was supplemented with additional 3.8 ml lysis buffer and incubated on ice for 25 min. Lysis efficiency was assessed using an optical microscope. After incubation, a 30 µm strainer was used to filter the lysate and washed with 1 ml lysis buffer. The lysate was centrifuged at 500 g (5 min, 4°C) and the supernatant was discarded. 5 ml washing buffer (0.01% BSA, 0.4 U/µl SUPERaseIN RNAse Inhibitor and 0.4 U/µl RNasin® Plus Ribonuclease Inhibitor in PBS) were added to the pellet and transferred to a falcon tube coated with 0.1% BSA in PBS and centrifuged (500 g, 5 min, 4°C). Resuspension buffer (1% BSA and 0.4 U/µl RNasin® Plus Ribonuclease Inhibitor in PBS) was added to the pellet. 40,000 nuclei were loaded onto a Chip G for GEM generation using the Chromium Single Cell 3' v3.1 kit (10X Genomics). Reverse transcription, barcoding, complementary DNA amplification and purification for library preparation were performed as specified in the manufacturer's protocol. Sequencing was performed on a NovaSeq 6000 platform (Illumina) at a target read depth of 25,000 by Novogene. libaries were constructed according to manufacturer specifications