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Sample GSM4450664

Query DataSets for GSM4450664

Status

Public on Apr 02, 2020

Title

CD133_1

Sample type

SRA

Source name

E13.5 embryonic DRG neuron culture

Organism

Mus musculus

Characteristics

tag: E13.5
lentivirus: human PROM1 overexpression
genotype/variation: CD133

Treatment protocol

For lentiviral transduction, lentivirus was added to the culture at DIV 2. For re-plating assay using the embryonic DRG cultures, neurons were re-plated at DIV 5. Briefly, cultures were incubated in DMEM/0.05% Trypsin-EDTA mixture (1:1) for 5 min at 37°C, 5% CO2 and rinsed with culture medium described above. Cells were then dissociated by gentle pipetting and transferred to new culture plates coated with poly-D-lysine/laminin. Re-plated Neurons were incubated over night at 37°C with 5% CO2.

Growth protocol

Embryonic DRG cultures were generated as previously described. Embryonic DRG collected from embryonic day 13.5 mice were dissociated in 0.05% trypsin-EDTA and plated on poly-D-lysine/laminin-coated dishes in Neurobasal medium (Gibco) supplemented with 2% B-27 (Gibco), 1% Glutamax, 1 μM 5-fluoro-2'-deoxyuridine (Sigma), 1 μM uridine (Sigma), 1% penicillin-streptomycin, and 50 ng/ml 2.5S nerve growth factor (Envigo, BT-5017).

Extracted molecule

total RNA

Extraction protocol

RNA extraction and analysis procedures were performed with RNAqueous Total RNA isolation kit (Thermo, AM1931), RevertAid Reverse Transcriptase (Thermo, EP0441), and PowerUP SYBR green master mix (Thermo, A25918) from L4-5 DRG tissues or cultured embryonic neurons (E13, DIV 5), following the manufacturer’s instructions. The transcript levels were compared between conditions by the ΔΔCt method, using Gapdh transcript levels as an internal control.
RNA-Seq libraries were generated by using strand-displacement stop/ligation method from cultured embryonic DRG neurons (E13) transduced with either control or human PROM1-overexpressing lentivirus. From 1.5μg of large RNA (>200nt, extracted by RNeasy Mini Kit; Qiagen), polyadenylated mRNA was purified by using 10μl of Dynabead Oligo(dT)25 (Invitrogen) according to protocol provided by manufacturer. Then, 10ng of the purified mRNA was subjected to prepare RNA-Seq library by using SENSE Total RNA-Seq Library Kit (Lexogen) as instructed by manufacturer. After the libraries were produced, they were amplified by PCR with the lowest optimal cycle, which was determined by comparing results from different cycles. The quality of the amplified library was checked by using Fragment Analyzer (Advanced analytical) for its size distribution and quantity. Finally, the prepared multiplexed library was precisely quantitated by using both Qubit RNA HS assay kit (Thermo Fisher) and Fragment Analyzer, applied to be sequenced by HiSeq 2500 system (Illumina) as 50 single-end reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

mDRG(cont_vs_CD133)_RNA-Seq_CHI_092818.xlsx

Data processing

The de-multiplexed sequencing reads (obtained by using CASAVA; Illumina) were aligned to the mouse genome (mm9) using TopHat2 (tophat2 -a 4 -g 1 --b2-sensitive -r 100 --mate-std-dev=50 --library-type fr-firststrand) under a supply of RefSeq gene annotations. (Huang et al., 2009a,b). Enriched biological process terms (P<0.001) were visualized by using REVIGO (http://revigo.irb.hr/) in semantic space as cluster representatives, after reduction of the redundant terms. Functional protein association was analyzed by STRING (https://string-db.org/).
Genome_build: mm9
Supplementary_files_format_and_content: Excel file, Differential expression list

Submission date

Apr 01, 2020

Last update date

Apr 03, 2020

Contact name

Yongcheol Cho

E-mail(s)

cry4free1@gmail.com

Phone

82-53-785-6190

Organization name

DGIST