Summary: The 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.
Overall Design: 26 Llive and 19 Fixed cultured hESCs were prepared and sequenced using both FRISCR and TritonX-100 Lysis as proof of principal for FRSCR.
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Growth Protocol: | H1 hESC’s (WiCell, Madison WI) were maintained on Matrigel (Corning) in mTESR1 media (StemCell Technologies, Vancouver BC). Adherent cell cultures were dissociated with StemPro Accutase Cell Dissociation Reagent (Life Technologies, Chicago IL). The cells were centrifuged (220xg, 3 min) and the dissociation solution was removed. |
Treatment Protocol: | - |
Extract Protocol: | Cortical 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 Protocol: | Library construction was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit. |
Molecule Type: | poly(A)+ RNA |
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Library Layout: | PAIRED |
Library Strand: | - |
Platform: | ILLUMINA |
Instrument Model: | Illumina HiSeq 2500 |
Strand-Specific: | Unspecific |
Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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