Computational protocol: Repulsive cues combined with physical barriers and cell–cell adhesion determine progenitor cell positioning during organogenesis

Similar protocols

Protocol publication

[…] Whole-mount in situ hybridization was performed according to ref. , with the detailed protocol provided in . Digoxigenin and fluorescein-labelled probes for chromogenic in situ were synthesized according to the manufacturer's protocol (Roche, Switzerland). Images of Chromogenic WISH and histological sections were captured on an Axioplan2 microscope (Zeiss) using a SPOT Xplorer camera (SPOT Imaging Solutions) controlled by either Metamorph (Visitron Systems) or SPOT (SPOT Imaging Solutions) software and processed using ImageJ (NIH). RNAscope procedure was performed according to ref. , with the detailed protocol provided in and imaged as z-stacks of whole embryos using a Z.1 light-sheet microscope (Zeiss) controlled by ZEN software (Zeiss) followed by image processing using Imaris software (Bitplane, Switzerland; ; ). A list of the probes and the primers used to amplify them is provided in . [...] PGCs from 7 and 36 hpf embryos carrying the kop-egfp-f-nos3′ transgene were sorted using FACS (FACSAria cell sorter (BD Biosciences) equipped with a 70 μm nozzle). 500 ng of total RNA from PGCs and somatic cells was isolated using the PicoPure RNA extraction kit (Arcturus; Alphametrix). Generation of the RNA-seq library and Illumina-based high-throughput sequencing were performed in the Microarray Hybridization and Analysis Core of the Johns Hopkins University (USA). 7 hpf samples were sequenced on GAII platform, with ∼25 million paired reads per sample (2X75); 36 hpf samples were sequenced on HiSeq 2000 platform, with 150 million paired reads per sample (2X100). The reads were filtered and trimmed for low-quality reads and then mapped to the transcriptome using SeqMap (Bowtie for the last two samples) and generated expression value using rSeq. Differential expression was detected using DEseq package of Bioconductor. None of the transcripts were excluded. The expression levels of LPA and S1P receptors, sox32 RNA and cell adhesion molecules are presented in . […]

Pipeline specifications

Software tools Bowtie, DESeq
Application RNA-seq analysis