Dataset View [GSE71858]

TitleFixed single-cell transcriptomic characterization of human radial glial diversity
ArticleRamanathan S,Levi BP,Shapovalova NV,Nelson AM,Shehata SI,Jang S,Doyle AM,Hodge RD,Yao Z,Mich JK,Thomsen ER.Fixed single-cell transcriptomic characterization of human radial glial diversity.Nature methods.2016 Jan
Bio ProjectBioProject: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA292324
SraSRA: http://www.ncbi.nlm.nih.gov/sra?term=SRP062177
Overall Desgin26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR.
SummaryThe human neocortex is created from diverse progenitors that are intermixed with multiple cell types in the prenatal germinal zones. These progenitors have been difficult to profile with unbiased transcriptomics since progenitors-particularly radial glia (RG)-are rare cell types, defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed a method called FRSCR for transcriptome profiling of individual fixed, stained, and sorted cells. After validation of FRSCR with human embryonic stem cells, we profiled primary human RG that constitute only 1% of the mid-gestation cortex. These data showed that RG could be classified into ventricle zone-enriched RG (vRG) that expressed ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that expressed HOPX. Our study identified the first markers and molecular profiles of vRG and oRG cells, and provides an essential step for understanding molecular networks that control the development and lineage of human neocortical progenitors. Furthermore, FRSCR allows targeted single-cell transcriptomic profiling of many tissues that currently lack live-cell markers.
Experimental ProtocolCortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4oC, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca2+- and Mg2+-free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL). Minced pieces were treated with 2 mL trypsin solution for 20 min at 37 oC ( Ca2+- and Mg2+-free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 μg/mL DNase I (Roche, Basel, Switzerland), pH 7.6). Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 mL 1M HEPES pH 7.3–7.4, 5 ml 100x Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na2H2EDTA], 1g bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 μg/mL trypsin inhibitor (T6522, Sigma) and 10 μg/mL DNase I (Roche). Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles. The cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically ~30-50 million live cells isolated per cortical piece at ~50% viability).; Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit.
Data processingRaw read data was aligned to GRCh37 (hg19) using the RefSeq annotation gff file downloaded on 4/23/2013. Transcriptome alignment was performed first using RSEM39, unmapped reads were then aligned to hg19 using Bowtie40, and remaining unmapped reads were aligned to the ERCC sequences.; Genome_build: hg19; Supplementary_files_format_and_content: Gene counts and normalized TPM (transcripts per milllion reads) were provided for each single cell sample
Public OnPublic on Oct 02 2015

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