title: GSM3267148: BMP GFP_1; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA from transfected and FACS sorted cells was extracted using Qiagen RNAeasy Micro kit (Qiagen, 74104) . Total RNA from non-transfected cells was extracted using Direct-zol™ RNA MiniPrep Kit (Zymo Research, R2052), according to manufacturer's instructions. RNA concentration was quantified using the Qubit dsDNA BR/HS Assay Kit. A KAPA mRNA HyperPrep Kit (for Illumina) (KAPA Biosystems, Wilmington, MA, USA) has been used with 1000ng of RNA diluted to a final volume of 50µl. Each RNA sample was captured with 50µl of capture beads at 65°C for 2 min and 20°C for 5 min. For the second capture, 50µl of RNase free water was used at 70°C for 2 min and 20°C for 5 min. Captured RNA was subjected to the KAPA Hyper Prep assay: end-repair, A-tailing, and ligation by adding 11µl of Fragment, Prime and Elite Buffer (2X). To obtain a distribution of 200-300bp fragment on the library, the reaction was run for 6min at 94°C. cDNA synthesis was run in 2 steps following manufacturer's instructions. The ligation step consisted of a final volume of 110 μL of the adaptor ligation reaction mixture with 60μL of input cDNA, 5 μL of diluted adaptor and 45μL of ligation mix (50µL of ligation buffer+ 10 μL of DNA ligase). The Kapa Dual-Indexed Adapters (KAPA Biosystems-KK8720) stock was diluted to 7µM (1.5mM or 7nM) to get the best adaptor concentration for library construction. The ligation cycle was run according to manufacturer's instructions. To remove short fragments such as adapter dimers, 2 AMPure XP bead clean-ups were done (0.63 SPRI and 0.7SPRI). To amplify the library, 7 PCR cycles were applied to cDNA KAPA HP mix. Amplified libraries were purified using AMPure XP. The quality and fragment size distributions of the purified libraries was assessed by a 2200 TapeStation Instrument (Agilent Technologies, Santa Clara, CA, USA).