title: GSM5739218: E12 cKO G rep 2; Mus musculus; RNA-Seq
library_construction_protocol: After cell sorting of E13/E16 and P0 cells, samples were incubated for 30 min at 37 °C and stored at -80°C until further usage. After thawing samples on ice, their total volume was adjusted to 250 µl using RNase-free H2O (Thermo Fisher Scientific) followed by addition of 750 µl Trizol LS (Thermo Fisher Scientific) and inverting five times. After incubating for 5-min at RT, the entire solution was transferred into a MaXtract tube (Qiagen). 200µl microliters chloroform (Sigma-Aldrich) were added. After three times 5sec vortexing and 2min incubation at RT, samples were centrifuged for 2 min with 12,000 rpm 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, 1 µl GlycoBlue (Thermo Fisher Scientific) was added and entire solution was mixed by vortexing 3 × 5sec. Samples were incubated at -20°C for 3 nights to precipitate RNA. Then, samples were centrifuged for 20min with 14,000 rpm at 4 °C. Supernatant was removed and RNA pellet was washed with 70% ethanol, followed by a 5min centrifugation step (14,000 rpm at 4 °C). RNA pellet was resuspended in 12.5 µl RNase-free H2O. RNA quality was analyzed using Bioanalyzer RNA 6000 Pico kit (Agilent) following the manufacturer's instructions. RNA samples were pipetted into a 96well plate, sealed with AluminumSeal and stored at −80 °C until further use. Sequencing libraries were prepared following the Smart-Seq v2 protocol (Picelli et al., 2013) using custom reagents (VBCF GmbH) and libraries from a 96-well plate were pooled, diluted and sequenced on a HiSeq 2500 (Illumina)