ena-STUDY-DPSQ-14-03-2018-19:09:10:340-151
The transcriptome of Gfi1aa and/or Gfi1b-depleted primitive red blood cells in zebrafish embryos
The zebrafish gene trap line qmc551 carries a gene trap transposon in intron 1 of the gene gfi1aa which a encodes a transcriptional repressor. The presence of the transposon interferes with the transcription of the gfi1aa gene during haematopoiesis. Despite the lack of Gfi1aa expression, primitive red blood cell development appears to be normal due to functional redundancy with a second gene called gfi1b. Loss of Gfi1aa and Gfi1b protein expression in embryos that are homozygous for qmc551 and injected with gfi1b morpholinos leads to maturation defects in primitive red blood cells. Here, we performed an RNA-sequencing experiment to study the early programming of Gfi1aa and/or Gfi1b-depleted primitive red blood cells. For this purpose, we made use of the gene trap's gfp reporter gene expression in the primitive red blood cells to isolate these cells by fluorescence-activated cell sorting from either qmc551 heterozygous or homozygous 20-hour-old embryos that were or were not injected with the gfi1b morpholinos at the one-cell stage. Batches of 200 embryos were dissociated in a Liberase Blendzyme solution. GFP-positive primitive red blood cells were isolated by fluorescence-activated cell sorting. 3 samples of between 1700 and 4000 cells were collected per batch. Total RNA was isolated from the cells of each of the 4 x 3 samples (Het1-3, HetMO1-3, Hom1-3 and HomMO1-3) and used to generate full-length cDNA of polyadenylated transcripts. Following cDNA amplification and library preparation paired end 75 bp reads were generated on the Illumina NextSeq500 sequencing platform.
The zebrafish gene trap line qmc551 carries a gene trap transposon in intron 1 of the gene gfi1aa which a encodes a transcriptional repressor. The presence of the transposon interferes with the transcription of the gfi1aa gene during haematopoiesis. Despite the lack of Gfi1aa expression, primitive red blood cell development appears to be normal due to functional redundancy with a second gene called gfi1b. Loss of Gfi1aa and Gfi1b protein expression in embryos that are homozygous for qmc551 and injected with gfi1b morpholinos leads to maturation defects in primitive red blood cells. Here, we performed an RNA-sequencing experiment to study the early programming of Gfi1aa and/or Gfi1b-depleted primitive red blood cells. For this purpose, we made use of the gene trap's gfp reporter gene expression in the primitive red blood cells to isolate these cells by fluorescence-activated cell sorting from either qmc551 heterozygous or homozygous 20-hour-old embryos that were or were not injected with the gfi1b morpholinos at the one-cell stage. Batches of 200 embryos were dissociated in a Liberase Blendzyme solution. GFP-positive primitive red blood cells were isolated by fluorescence-activated cell sorting. 3 samples of between 1700 and 4000 cells were collected per batch. Total RNA was isolated from the cells of each of the 4 x 3 samples (Het1-3, HetMO1-3, Hom1-3 and HomMO1-3) and used to generate full-length cDNA of polyadenylated transcripts. Following cDNA amplification and library preparation paired end 75 bp reads were generated on the Illumina NextSeq500 sequencing platform.