GSM2360212: RVKB019C_dotflaA_Myd88; Mus musculus; RNA-Seq
Ribosome profiling experiments were undertaken essentially as previously described (Ingolia et al., 2012, PMID: 22836135). For all experiments cells were lysed by flash freezing and thawed in the presence of lysis buffer as previously described (Ingolia et al., 2012, PMID: 22836135). Clarified lysates were split and some was used to generate ribosome footprints while some was used to isolate total RNA for RNA sequencing. For RNAseq libraries 300μL of clarified lysate was mixed with 900μL of Trizol LS (Life Technologies) and RNA was isolated following the manufacturer’s guidelines. Ribosome profiling libraries were generated as previously described (Ingolia et al., 2012, PMID: 22836135). All RNA and DNA gel extractions were performed overnight as previously described (Ingolia et al., 2012, PMID: 22836135). The Ribo-Zero Gold rRNA Removal Kit (Illumina) was used to remove rRNA from ribosome profiling samples before the dephosphorylation and linker ligation steps described (Ingolia et al., 2012, PMID: 22836135). For RNAseq libraries total RNA was submitted to the University of California, Berkeley Functional Genomics Laboratory. There the Ribo-Zero Gold rRNA Removal Kit (Illumina) was used to remove rRNA. RNA integrity was measured utilizing the RNA Pico method on the Agilent 2100 Bioanalyzer at the University of California, Berkeley Functional Genomics Laboratory. High quality RNA with a RNA integrity number (RIN) >8 (Agilent Technologies) was used to generate libraries following standard Illumina protocols for single read 100 (RS100) sequencing. Final ribosome profiling libraries were sequenced on a HiSeq2000 System (Illumina) with single read 50 (SR50) read lengths and final RNAseq libraries were sequenced on a HiSeq2000 System (Illumina) with SR100 read lengths by the Vincent J. Coates Genomics Sequencing Laboratory at UC, Berkeley.
GEO Accession
GSM2360212