GSM1201482: RNA in MEFs derived from JF1 (mother) X TgOG2 (father) cross; Mus musculus; RNA-Seq
RNA was isolated using RNA-Bee (AMS Biotechnology). DNAseI digestion was applied to remove DNA contamination. 10 μg total RNA was treated with the Ribominus kit (Life Technologies) to remove ribosomal RNA contamination. The amount of RNA was monitored using a Nanodrop spectrophotometer followed by a quality check using the Bioanalyzer. First-strand synthesis was done using Superscript II reverse transcriptase, dNTPs and the Illumina RNA seq library preparation kit. The enzyme was inactivated and the dNTPs were removed. To enable strand-specific analysis, the second strand cDNA was labeled with dUTP before ligating the adapter. The second strand was synthesized using E. coli DNA polymerase I and dATP, dGTP, dCTP, dUTP nucleotides in the presence of E. coli ligase and RNase H. Ends were repaired using “End It Enzyme mix”, and dATP 3’ overhangs were created using Klenow exo- and then R2 adapters were ligated to the double stranded cDNA fragments. The second strand was degraded using USER™ (Uracil-Specific Excision Reagent) enzyme complex. Only the first strand was used as PCR template for paired-end deep sequencing. The read1 sequences (F2R1 or R1F2) represent the antisense strand and the read 2 (F1R2 or R2F1) sequences represent the sense strand of the RNA. The single stranded cDNA containing the adapter was amplified by PCR using two different index primers. Reads resulting from the JXO cross MEF started with the sequence ACTTGA and reads resulting from the OXJ cross MEF started with the sequence CCGTCC. The two MEF samples were sequenced on half a lane each of the Illumina sequencer.