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Pipeline publication

[…] ecutive PBS washes, transcription inhibition was released by the addition of fresh complete medium. Every 5 min, one well was washed with PBS and directly lysed in TRIzol reagent (Thermo Fisher, 15596026) at −80°C. Total RNA was isolated following the instructions for the TRIzol reagent. 500 ng of total RNA was used for cDNA synthesis with the Superscript II RT enzyme (Thermo Fisher, 18064014) and random hexamer primers (Roche, 11034731001) following the manufacturer's instructions. RT–qPCRs were performed in triplicates in 20 μl total volume in a Bio‐Rad CFX Connect real‐time PCR detection system with GoTaq qPCR master‐mix (Promega, A6001). We used the C t threshold cycle determined by the CFX Manager software in accordance with the 2−ΔΔCt method to analyze RT–qPCR data. The ratio relative to the untreated control sample is plotted., mRNAseq: Adapters were trimmed from the 3′ ends of the reads with cutadapt v1.10 (Martin ). The trimmed reads larger than 18 nt were aligned against the rDNA of the respective organism using bowtie2 v 2.1.0 (Langmead and Salzberg, ; –sensitive‐local –score‐min G,30,8 ‐N 0 –ignore‐quals) to remove the rRNA reads. The rRNA cleaned reads were aligned to the genome with the TopHat splice junction mapper for RNA‐seq reads v1.4.1 (Trapnell et al, )., ChIPseq: Adapters were trimmed from the 3′ ends of the reads with cutadapt v1.10. The trimmed reads larger than 18 nt were aligned to the genome (NCBI mm9) using bowtie v1.0.0 (Langmead et al, ; ‐v 2 –best –strata –tryhard ‐m 1)., GROseq: Adapters were trimmed from the 3′ ends of the reads with cutadapt (v 1.10), and a 5‐nt random linker was removed from the 3′ end using fastx_trimmer. Trimmed reads larger than 10 nt were aligned to the genome (NCBI mm9) using bowtie v1.0.0 (‐v 2 –best –strata –tryhard ‐m 1)., All bioinformatics analyses were based on gene annotation from the RefSeq database (downloaded from UCSC on […]

Pipeline specifications

Software tools CFX Manager, cutadapt, Bowtie2, TopHat