Fetal liver erythroblasts from E11.5 embryos were stained with Zombie NIR Fixable Viability Kit (Biolegend) to discriminate dead cells, followed by the staining with anti-CD71 and anti-Ter119 antibodies (Biolegend). Live CD71+Ter119+ and CD71+Ter119- cells were sorted using an SH800Z cell sorter. The total RNA of sorted cells was isolated using NucleoSpin RNA XS (Takara Bio) according to the manufacturer’s protocol.
RNA integrity (RIN) was examined using Bioanalyzer (Agilent) with RNA 6000 Pico Kit (Agilent) to confirm the RIN > 7. Then, the RNA-seq libraries for Illumina sequencing were generated using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs) according to the manufacturer’s protocol. Sequencing was performed using NextSeq 500 Sequencer and NextSeq 500/550 High-Output v2 Kit (Illumina).
The RNA-seq analysis was performed using the Galaxy web server (use.galaxy.org). Raw reads were trimmed by cutadapt (http://journal.embnet.org/index.php/embnetjournal/article/view/200 ), aligned to the mm10 murine reference genome by HISAT284 (link), and then counted via the featureCounts package85 (link). The read counts were further processed by limma86 (link).
RNA integrity (RIN) was examined using Bioanalyzer (Agilent) with RNA 6000 Pico Kit (Agilent) to confirm the RIN > 7. Then, the RNA-seq libraries for Illumina sequencing were generated using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs) according to the manufacturer’s protocol. Sequencing was performed using NextSeq 500 Sequencer and NextSeq 500/550 High-Output v2 Kit (Illumina).
The RNA-seq analysis was performed using the Galaxy web server (use.galaxy.org). Raw reads were trimmed by cutadapt (
Free full text: Click here
