GSM4103096: Neuron replicate 1; Mus musculus; OTHER
Upon cell sorting, samples were incubated for 30min at 37°C. Total volume was adjusted to 250µl using RNase-free H2O (Thermo Fisher Scientific) followed by addition of 750µl Trizol LS (Thermo Fisher Scientific). Samples were mixed by inverting 5 times. After a 5min incubation step at RT, the entire solution was transferred into a MaXtract tube (Qiagen). 200µl chloroform (Sigma-Aldrich) were added, followed by 3 times 5sec vortexing and 2min incubation at RT. Samples were centrifuged for 2min with 12000rpm at 18°C. Supernatant was transferred to a new tube and isopropanol (Sigma-Aldrich) was added in a 1:1 ratio. For better visibility of the RNA pellet 1µl GlycoBlue (Thermo Fisher Scientific) was added and entire solution was mixed by vortexing (3x 5sec). Samples were left for precipitation o/n at -20°C. After precipitation samples were centrifuged for 20min with 14000rpm at 4°C. Supernatant was removed and RNA pellet was washed with 70% ethanol, followed by a 5min centrifugation step (14000rpm at 4°C). RNA pellet was resuspended in 12,5µl RNase-free H2O. RNA quality was analyzed using Bioanalyzer RNA 6000 Pico kit (Agilent) following the manufacturer's instructions. RNA samples were stored at -80°C until further use. RNA was isolated from 10.000 sorted cells using Trizol LS (Thermo Fisher Scientific), DNAse1 treated (DNA free KIT, Ambion) and converted to cDNA (SuperScript VILO, Invitrogen). Genomic DNA was prepared from the carcass of one embryo used for FACS sorting. For deep sequencing, regions of interest were amplified with different primers that produce fragments where the SNP of interest is located within 50bp of one end, with a high fidelity DNA polymerase. PCR fragments were purified (Ampure XP, Beckman Coulter) and pooled per sample of origin. Each pool was prepared for deep sequencing (VBCF GmbH, Vienna) and sequenced on a HiSeq 2500 (Illumina) using v4 chemistry. targeted sequencing of cDNA