RNA level differences between neurons in soft and stiff environments
RNA level differences between neurons in soft and stiff environments
Primary hippocampal cells from rat embryos were grown in gels of different stiffnesses (10kpa and 100pa) attached to ibidi 35mm diameter glass bottom dishes. 2 separate CRISPR assays in parallel (H1 and H2). Each of those assays has been carried out by electroporating 4 different CRISPR guides into the cells per assay. All 8 guides are specific for the PIEZO1 gene. TOPO cloning with subsequent Sanger sequencing has shown random variations for the different guides in the guide regions (or close to it). But the efficiency of the KD is low. We estimate between 25% and 40%. For RNA isolation culture medium was taken off and 350ul RLT plus buffer containing beta mercapto ethanol was added to the gel containing the cells. The gel containing the cells was scraped off the dish (Cell Scraper Greiner Bio one), transferred to a 1.5 ml Eppendorf and then homogenized by pipetting up and down (x50) using a blue tip cut off to increase the tip opening. Each lysate was then vortexed 30 sec. RNA purification was performed as described by RNeasy plus Micro kit (QIAGEN #74034). Samples were eluted in 20ul of RNase-free H2O and quantified measuring UV absorption at 260 nm with a spectrophotometer plate reader, the SpectroStar (BMG Labtech).  Libraries were generated using the the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech Laboratories)