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Sample GSM1846992 Query DataSets for GSM1846992
Status Public on Oct 02, 2015
Title Live_PLRC [US-1457351-8]
Sample type SRA
 
Source name H1 ESCs p40 Live Cells FRSCR
Organism Homo sapiens
Characteristics cell line: H1
cell type: cultured embryonic stem cells
phenotype: DAPI-
sample group: Live cells_FRSCR
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.
Extracted molecule total RNA
Extraction 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 was carried out as previously reported by Smart-Seq2 and Nextera XT DNA prep kit.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Data processing Raw 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
 
Submission date Aug 07, 2015
Last update date May 15, 2019
Contact name Zizhen yao
E-mail(s) zizheny@alleninstitute.org
Organization name Allen Institute for Brain Science
Department MAT
Street address 551 N 34th St #200
City Seattle
State/province WA
ZIP/Postal code 98103
Country USA
 
Platform ID GPL16791
Series (1)
GSE71858 Fixed single-cell transcriptomic characterization of human radial glial diversity
Relations
SRA SRX1137136
BioSample SAMN03979816

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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