GSM8338112: Isthmus, rep3; Homo sapiens; RNA-Seq
Centrifuge cells at 300 rcf for 5 min at 4°C. Remove the supernatant without disturbing the cell pellet. Using a wide-bore pipette tip, add 200 ul Lysis Buffer  and gently pipette mix 5x or until cells are completely  suspended without introducing bubbles. Lyse the cells on ice for 1 min.  Using a regular-bore pipette tip, add 800 ul Nuclei Wash & Resuspension Buffer. Gently pipette mix 5x. and wash nuclei two times. Remove the supernatant without disrupting the pellet.  Using a regular-bore pipette tip, add 1 ml or an appropriate  volume Nuclei Wash & Resuspension Buffer to achieve a  target concentration of 700-1,200 nuclei/μl. Gently pipette  mix 10x or until nuclei are completely suspended.   Determine the nuclei concentration using a Countess II FL  Automated Cell Counter. Proceed immediately with the 10x Genomics Single  Cell protocol and minimize the time between nuclei  preparation and chip loading Preparation of single nuclei from frozen human HGSOC and fallopian tube samples and constructions of 3' single-cell gene expression libraries were conducted using the 10x Genomics Chromium system (Next GEM v3.1) following the manufacturer's protocol. The libraries were sequenced with ~200 million PE150 reads per sample on Illumina NovaSeq at LC-BIO Co., Ltd (Hangzhou, China).