GSM2200068: RP 4h_rep2; Mus musculus; RNA-Seq
RNA was isolated using QIAzol Lysis Reagent. Sample preparation for 4sU-RNA and total RNA was performed as described before (Rädle et al., 2013) with minor changes. In step 5 we eluted the newly transcribed RNA into 400 µl Agencourt RNAClean XP Beads (Beckman Coulter) and continued with the recovery following the manufacturer’s instructions. RNA was eluted in 11 µl nuclease-free H2O and quantified using Qubit 2.0 Fluorometer. Samples for ribosome profiling were prepared according to manufacturer’s instructions (Illumina, TrueSeq Ribo Profile, Mammalian). At least 60 x 10e6 Th1 cells were used for each time point. For both total and 4sU-RNA samples, library preparation and rRNA depletion was performed using the TruSeq® Stranded Total RNA Library Prep Kit (Illumina) starting with 400 ng RNA as input for each sample. Only 11 cycles were used for PCR amplification to minimize PCR bias. Libraries for ribosome profiling were prepared according to manufacturer’s instructions (Illumina, TrueSeq Ribo Profile, Mammalian) except that we used the whole amount of RNA for both gel purifications steps. Overnight gel extraction was used at every stage of the procedure. Depletion of rRNA was performed with Ribo-Zero Gold rRNA Removal Kit (Illumina, Human/Mouse/Rat) and RNA purification was done with RNA clean and concentrator kit according to the manufacurer’s advice (Zymo Research).
GEO
GSM2200068