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Protocol publication

[…] Total RNA was purified from fetal livers or sorted cell populations using TRIzol (Invitrogen). Complementary DNA (cDNA) was synthesized by Moloney murine leukemia virus reverse transcription (MMLV RT). Real-time PCR was performed with SYBR Green Master Mix. Control reactions lacking MMLV RT yielded little to no signal. Relative expression was determined from a standard curve of serial dilutions of cDNA samples, and values were normalized to 18S RNA expression. The specific primers used for real-time PCR are given in table S1. RNA-seq was conducted with an Illumina HiSeq 2000 system. Transcript quantification and differential expression were conducted using the software packages RSEM () and DESeq2 (), respectively. RSEM v1.2.18 was provided with a reference transcript set consisting of all protein-coding and large intergenic noncoding transcripts from the Ensembl release 67 annotation of the National Center for Biotechnology Information (NCBI) Build 38 mouse genome assembly. Default parameters and Bowtie v1.1.1 were used for transcript quantification with RSEM. Transcript read counts were summed to produce counts at the level of gene symbols. These counts were then given as inputs to DESeq2 v1.5.90 for differential expression analysis. DESeq2 was run with default parameters except for betaprior = F and alpha = 0.05. Gene symbols with Benjamini-Hochberg FDR values <0.05 were deemed to be differentially expressed between mutant and wild type. The accession number for the RNA-seq data is GEO: GSE69786. […]

Pipeline specifications

Software tools RSEM, DESeq2, Bowtie
Application RNA-seq analysis
Organisms Mus musculus
Diseases Anemia, Immunologic Deficiency Syndromes, Myelodysplastic Syndromes, Leukemia, Myeloid, Acute