Comment[GEAAccession]	E-GEAD-348
MAGE-TAB Version	1.1
Investigation Title	single cell analysis of wild type and Fbl mutant cells for dorsal brains
Experiment Description	"\"\"\"\"\"\"To isolated Hes1 positive neural stem cells (NSCs) at E14, dorsal cortices were dissected from Hes1-d2-EGFP Tg/+ mice. Cortices were dissociated with 0.05% trypsin with Hanks Balanced Salt Solution (HBSS) (-) at 37 degree for 10 min. After centrifugation at 1000g for 5 min, cells were re-suspended with 0.375% BSA/HBSS(-) by gentle pipetting 15 to 20 times. Re-suspended cells were filtered with 35 um filter (Falcon) and sorted into sorting buffer (20 ng/ml human basic FGF (Peprotech), 1XB27 RA- (Gibco), in Dulbeccos Modified Eagle Medium (DMEM) F12+GlutaMax (Gibco)) by a cell sorter (SH800, SONY) equipped with 130 um sorting chips (SONY, LE-C3113). After sorting, cell number was counted by Countess or Countess II (Invitrogen). For cells from wild type and Fbl mutant mice, cells were collected similarly except that sorting was not performed. 
Collected cells were immediately load into the 10X-Genomics Chromium (10X Genomics, Pleasanton, CA). Libraries for single cell cDNA were prepared using Chromium 3 v2 platform as the manufacturer's protocol.\"\"\"\"\"\""
Experimental Design	time series design	genotyping design	self vs self design
Experimental Factor Name	genotype
Experimental Factor Type	genotype
Person Last Name	wu
Person First Name	quan
Person Affiliation	RIKEN Center for Biosystems Dynamics Research
Person Roles	submitter
Public Release Date	2021-12-15
Protocol Name	P-GEAD-390	P-GEAD-391	P-GEAD-392	P-GEAD-393	P-GEAD-394
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid library construction protocol	nucleic acid sequencing protocol	normalization data transformation protocol
Protocol Description	All cells were from dorsal brains of wild type and Fbl mutant mice.	by gentle pipetting 15 to 20 times. Re-suspended cells were filtered with 35 um filter (Falcon) and sorted into sorting buffer (20 ng/ml human basic FGF (Peprotech), 1XB27 RA- (Gibco), in Dulbeccos Modified Eagle Medium (DMEM) F12+GlutaMax (Gibco)) by a cell sorter (SH800, SONY) equipped with 130 um sorting chips (SONY, LE-C3113). After sorting, cell number was counted by Countess or Countess II (Invitrogen). For cells from wild type and Fbl mutant mice, cells were collected similarly except that sorting was not performed.	Collected cells were immediately load into the 10X-Genomics Chromium (10X Genomics, Pleasanton, CA). Libraries for single cell cDNA were prepared using Chromium 3 v2 platform as the manufacturer protocol.	Sequencing was performed in HiSeq1500 (Illumina) with the the HiSeq PE Rapid Cluster Kit v2 (Illumina), or the TruSeq PE Cluster Kit v3-cBot-HS, to obtain paired-end 26 nt (Read 1)- 98 nt (Read 2) reads for scRNA-seq libraries	Sequenced data was mapped to mm10 and cell number and raw count for each gene were reported by cellranger 2.0.2 (10X GENOMICS). All data were further analyzed by a bioinformatics pipeline Seurat (2.3.4). Briefly, we first created a SeuratObject, in which genes that are expressed by less than 3 cells and cells that express less than 200 genes were removed. Number of unique molecular index (UMI) were automatically counted by Seurat. We calculated percentage of UMI mapping to mitochondrial genes and used these values to further filter cells (<15%). Data were then normalized ((normalization.method = \"LogNormalize\", scale.factor = 100000) and scaled (vars.to.repress=c(\"UMI\",\"percent.mito\")).
SDRF File	E-GEAD-348.sdrf.txt
Comment[AEExperimentType]	RNA-seq of coding RNA from single cells
Comment[BioProject]	PRJDB9278
Comment[Last Update Date]	2021-12-15