E-MTAB-6077
Determining single-cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hours post fertilization
Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer (10.7554/eLife.03848). Specifically, the 2.7 kb Mouse Smarcd3-F6 sequence (chr5: 24113559 -24116342 from mm9 assembly) was sub-cloned from a gateway entry vector into the Zebrafish Enhancer Detection (ZED). Tol2-mediated transgenesis was performed and stable lines were created. The Smarcd3-F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3-F6:EGFPhsc70 embryos were dissociated at 10 hours post fertilization for fluorescent activated cell sorting (FACS) to collect GFP+ cells. Single-cell cDNA libraries were prepared using Fluidgim C1 system ((Fluidgim, PN 100-7168 Rev. B1)). 96 single-cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells (number of genes detected > 2000) were kept for clustering analysis. For the tube control experiments we performed mRNA-seq on ~4000 cells from the same embryo batches as the scRNA-seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP- comparison.  Libraries were made following the Fluidgim C1 mRNA-seq tube control protocol (Fluidgim, PN 100-7168 Rev. B1). Polyadenylated mRNA was captured by Oligo-dT primers and PCR amplified after reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair-end sequenced on the Illumina HiSeq 2500 platform.
Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer (10.7554/eLife.03848). Specifically, the 2.7 kb Mouse Smarcd3-F6 sequence (chr5: 24113559 -24116342 from mm9 assembly) was sub-cloned from a gateway entry vector into the Zebrafish Enhancer Detection (ZED). Tol2-mediated transgenesis was performed and stable lines were created. The Smarcd3-F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3-F6:EGFPhsc70 embryos were dissociated at 10 hours post fertilization for fluorescent activated cell sorting (FACS) to collect GFP+ cells. Single-cell cDNA libraries were prepared using Fluidgim C1 system ((Fluidgim, PN 100-7168 Rev. B1)). 96 single-cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells (number of genes detected > 2000) were kept for clustering analysis. For the tube control experiments we performed mRNA-seq on ~4000 cells from the same embryo batches as the scRNA-seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP- comparison.  Libraries were made following the Fluidgim C1 mRNA-seq tube control protocol (Fluidgim, PN 100-7168 Rev. B1). Polyadenylated mRNA was captured by Oligo-dT primers and PCR amplified after reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair-end sequenced on the Illumina HiSeq 2500 platform.