Mechanical regulation of the electric maturation of hippocampal neurons
Mechanical regulation of the electric maturation of hippocampal neurons
The culture media was removed from the dishes. Cultured cells including the hydrogel substrates, were scraped off the dish and collected with a pipette. Pregnant rats were euthanised at E17/18. Uteri were removed and processed under sterile conditions. Embryonic sacs were opened, embryos were decapitated and brains were extracted in HBSS containing 1% PSF. Hippocampi were dissected in Hibernate E buffer 1% PSF and collected in 2 ml vials of Hibernate E + 1% PSF. Tissue was stored at 4°C overnight. E17-18 embryo, Tissue was washed 3 times in cold HBSS + 1% PSF and incubated with trypsin for 10 minutes at 37°C. Tissue was washed 10 times in warm HBSS + 1% PSF. Cells were then isolated by pipetting the tissue up and down in the Nucelofector solution and CRISPR-Cas9 reagents.This contained either the Cas9 protein and one of two sets of four different CRISPR-Cas9 guides (resulting in the two knockdown conditions H1 and H2) targeting the Piezo1 gene, or the Cas9 protein and the TRACR RNA (control, also referred to as HWT.).  The cell suspension was then electroporated using a LONZA Nucleofector. Cells were seeded onto hydrogels of varying stiffness. The media was completely changed after 4-8 hours. Thereafter, half of the media was replaced every 3 days. Align to Rnor_6.0 reference genome using star (version 2.6.1d)    Samples were processed according to the manual of the Qiagen RNeasy plus Micro Kit (# 74034). DNAse treated samples at concentrations up to 50ng/ μl were submitted to CGS (Cambridge Genome Services) for RNA QC, library preparation, library QC and sequencing (project reference number: NGS-K.Mooslehner-Allen-40654). CGS used the Clontech SmartSeqV4 kit for cDNA and library preparation from our samples. The kit used oligo(dT) priming to generate high quality, full length cDNA directly from 10 pg to 10 ng total RNA. In addition to the cDNA synthesis kit, the SSv4 PLUS kit included the library preparation kit and a single-use Unique Dual Index (UDI) plate to generate Illumina-compatible sequencing libraries.