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Sample GSM1846987

Query DataSets for GSM1846987

Status

Public on Oct 02, 2015

Title

Live_RLT [US-1457351-3]

Sample type

SRA

Source name

H1 ESCs p40 Live Cells Triton X Lysis

Organism

Homo sapiens

Characteristics

cell line: H1
cell type: cultured embryonic stem cells
phenotype: DAPI-
sample group: Live cells_Triton X Lysis

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