title: Illumina NovaSeq 6000 paired end sequencing; Single-cell RNA-Seq to study the effects of acute and chronic CENP-A overexpression depending on p53 status
design: Single-cell RNA-Seq to study the effects of acute and chronic CENP-A overexpression depending on p53 status
library_construction_protocol: We grew MCF10-2A TetOn-CENPA-FLAG-HA cells expressing either an empty control vector (p53-WT) or dominant-negative p53 (p53-DN) without Dox or with continuous exposure to Dox (10ng/ml, ++, chronic) for 69 days in parallel. Cells in the no Dox condition were split into two dishes at day 67 and Dox was added to one set on day 68 for 24h of CENP-A overexpression (10 ng/ml, +, acute) while the other remained without Dox (-, control). We prepared samples in singlet according to the 10x Genomics Sample Preparation Demonstrated Protocol. In brief, we harvested cells by trypsinization, resuspended in typical media and mixed thoroughly by pipette, passed cells through a 40µm cell strainer, and counted cells by Beckman Automated Cell Counter. We resuspended cells in 1x PBS + 0.04% BSA, mixed, centrifuged gently, aspired supernatant and repeated wash two times. We passed cells through another cell strainer (40µm), counted again and diluted to a final concentration between 700 and 1200 cells/µl. We then proceeded directly to GEM generation and barcoding using the Chromium Single-cell 3' Reagent Kits v2 protocol with a targeted cell recovery of 2000 cells per sample, followed by post GEM-RT cleanup and cDNA amplification. 3' Gene Expression Library Construction, according to the manufacturer's instructions.