GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM1077448

Query DataSets for GSM1077448

Status

Public on May 23, 2013

Title

CEC Young

Sample type

SRA

Source name

Pool of corneal endothelial cells from 5 young donors

Organism

Homo sapiens

Characteristics

tissue: corneal endothelial cells

Treatment protocol

Research corneas were incubated in three washes of antibiotic/antimycotic solution in PBS, 15 minutes each. Corneoscleral rims were placed endothelial-side-up on a disposable cornea vacuum punch (Ripon, England), and mildly stabilized by the vacuum suction created. A brief 30 seconds treatment with Trypan Blue solution (0.1%) was used to delineate Schwalbe’s line. The CEC-DM layer was carefully stripped off, approximately 1 mm anterior to the Schwalbe’s line (away from the trabecular meshwork) from the posterior stroma under the dissecting microscope (Nikon, Japan).

Growth protocol

Research corneas were preserved and transported in Optisol-GS at 4°C, and were used within 13 days from preservation. Culture of Human Corneal Endothelial Cells: CEC-DM layer was digested enzymatically in collagenase A (2 mg/ml) for at least 2 hours and up to 6 hours. This allowed full detachment of the CECs from the DM, which tended to conglomerate into tightly-packed CEC clusters. The CEC clusters were rinsed once in PBS and further dissociated in TrypLE Express (TE) for 5 minutes. Cell pellets collected after a mild centrifugation (800 g for 5 minutes) were plated in culture-ware coated with FNC coating mixture. Isolated cells were left to adhere overnight in a stabilization medium made up of Human Endothelial-SFM supplemented with 5% FBS and 1x anti-biotic/anti-mycotic. Adhered hCECs were then cultured in F99 medium containing Ham’s F12 and M199, mixed in a 1:1 ratio, supplemented with 5% FBS, 20 μg/ml ascorbic acid, 1x Insulin-Transferrin-Selenium, 1x anti-biotic/anti-mycotic and 10 ng/ml bFGF. When the cultured cells reached 80-90% confluence, they were re-exposed to the stabilization medium for at least one week before passaging. The inclusion of this final step enhanced the general morphology of cultured hCECs (unpublished observation; manuscript in preparation). Confluent hCECs were passaged using TE, and seeded onto FNC-coated culture ware at a plating density of approximately 10,000 cells per cm2 for subsequent passage. All incubation and cultivation of hCECs were carried out in a humidified incubator at 37°C containing 5% CO2 unless otherwise stated. Fresh media were replenished every two days.

Extracted molecule

total RNA

Extraction protocol

For direct RNA extraction of donor tissues, isolated CEC-DM and corresponding corneal stroma button, punched out from the donor cornea tissue using an 8.0-mm diameter trephine, were rinsed once in PBS and placed directly into 1ml Trizol reagent. It should be noted that although most of the corneal epithelium spontaneously sloughed off the cornea surface during the transport and process of the cornea, remnant cells of the basal corneal epithelium might still be present on the corneal stroma button used for RNA extraction. Tissues were homogenized using a hand-held homogenizer before addition of 200ul of chloroform. After vigorous shaking, samples were spun at 13000 rpm for 15min at 4°C. The upper phase, containing total RNA, was transferred to a new tube containing an equal volume of 70% ethanol. The resulting solution was transferred into a QIAGEN RNeasy column and procedures were performed as per manufacturer’s protocol with a DNAse digestion step incorporated.
Libraries were prepared using an AB Demonstrated Protocol similar to the one reported in Tang et al 2009. Starting amount of RNA was 100pg. SOLiD library preparation kit was used for library generation.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

AB SOLiD 4 System

Data processing

Reads were alighned with ABI Bioscope version 1.3. Reads were first filtered for rRNA and tRNA samples and then aligned to the human genome (hg19.)
The uniquely aligned read counts (wt.merge.score.zone=4) were used for the calculation of RPKM and RPM values based on RefSeq annotation.
Genome_build: hg19
Supplementary_files_format_and_content: RPKM values in text format.

Submission date

Feb 05, 2013

Last update date

May 15, 2019

Contact name

Zhenzhi Chng

E-mail(s)

zzchng@gmail.com

Organization name

Institute of Medical Biology

Lab

Alan Colman