title: GSM4671713: 35011: M7_Nkx_Cx_wt; Mus musculus; RNA-Seq
library_construction_protocol: Samples in isolation Buffer (IB; Lexogen) were processed according to the SPLIT kit instructions (Lexogen). All other samples were incubated for 30min at 37°C. Total volume was filled to 250µl using RNase-free H2O (Thermo Fisher Scientific) followed by addition of 750µl Trizol LS (Thermo Fisher Scientific). Samples were mixed by 5 times inverting. After a 5min incubation step at RT, the entire solution was transferred into a MaXtract tube (Qiagen). 200µl chloroform (Sigma-Aldrich) was 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 an appropriate amount of RNase-free H2O. RNA quality was analyzed using Bioanalyzer RNA 6000 Nano (Agilent) and RNA 6000 Pico kit (Agilent) following the manufacturer's instructions. cDNA libraries were prepared using QuantSeq 3'mRNA library preparation kit (Lexogen) following the manufacturer's instructions. In order to amplify libraries correctly RT-PCR was performed as indicated in the protocol using PCR Add-on kit (Lexogen). cDNA library size distribution was analyzed using Bioanalyzer DNA High Sensitivity kit (Agilent) according to the manufacturer's instructions. cDNA library concentration was measured using Qubit. Libraries were pooled according to library length and concentration.
identifiers.namespace: GEO
identifiers.id: GSM4671713