title: GSM4671158: FVB_Cast_Cx_whole_tissue_2; Mus musculus; RNA-Seq
library_construction_protocol: Whole tissue samples were processed according to the Trizol LS protocol. 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. 500ng of total RNA were treated with DNase1 (Applied Biosystems) and purified using Ampure RNAClean XP beads. 200ng of the purified RNA was used for library preparation using the Illumina TruSeq Stranded Total RNA LT - (with Ribo-Zero TM Gold) - according to the manufacturer's protocol. 7-40ng of total RNA from FACS sorted cells were used for library preparation with the Clonetech SMARTer Stranded Total RNA Sample Prep Kit – Low Input Mammalian (with Ribogone), according to the manufacturer's protocol with 19 cycles of library amplification. If the concentration of the total RNA sample was too low, RNA was immobilized on Agencourt RNAclean XP beads according to the manufacturer's protocol. After washing and drying of the beads the RNA was eluted in 5yl water and the whole volume (including beads) was used in the RiboGone reaction.
identifiers.namespace: GEO
identifiers.id: GSM4671158