Computational protocol: Generation and Analysis of GATA2w/eGFP Human ESCs Reveal ITGB3/CD61 as a Reliable Marker for Defining Hemogenic Endothelial Cells during Hematopoiesis

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Protocol publication

[…] RNA-seq and subsequent data analysis were conducted as described by . In brief, total RNA was isolated with a Direct-zol RNA MiniPrep kit (Zymo Research) and sequencing libraries was prepared with a TruSeq RNA Sample Prep Kit (Illumina) following the manufacturer's protocol. The samples were run on an MiSeq system with MiSeq Reagent Kits v2 (50 cycles) (Illumina). In particular, RNA-seq data were processed essentially as described by , reads were aligned to an index generated from the Ensembl transcriptome version 74 (hg19) using RSEM (v1.2.19), Bowtie2 (v2.2.5), and normalized with EDASeq (v2.2.0). Gene expression is expressed as “normalized tag count.” A threshold of at least 20 normalized tags in any condition was used to filter lowly expressed transcripts. Differential expression was performed using DESeq2 (v1.8.1) and genes were considered significant if they had a Benjamini-Hochberg corrected p value (q value) <0.1 and had a fold-change > 1.5. Gene ontology was performed using goseq (v1.20.0). Other analyses were performed using glbase (). […]

Pipeline specifications

Software tools RSEM, Bowtie2, EDASeq, DESeq2, GOseq, Glbase
Application RNA-seq analysis
Organisms Homo sapiens, Mus musculus