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Sample GSM5468166

Query DataSets for GSM5468166

Status

Public on Jul 23, 2021

Title

D105 RB1-Mut hESC (C3)-hRBOs rep1 RNA-seq

Sample type

SRA

Source name

human embryonic stem cells

Organism

Homo sapiens

Characteristics

organoids: human ES-derived retinoblastoma organoids
developmental/tumor stage: day 105
genotype: RB1 mutation knock-in hESCs

Growth protocol

Human retinal and Rb organoids differentiation were performed as previously described with minor modifications. On day 0, RB1Wt/Wt and RB1Mut/Mut hESCs maintained on Matrigel-coated plates were pretreated with 10 μM Y-27632 for 2h, and then carefully dissociated into single-cell suspensions using TrypLE Select (containing 0.05 mg/ml DNase I and 20 μM Y-27632), plated at a density of 12,000 live cells per well in 96-well low-attachment V-bottom plates in retinal differentiation medium I containing 44% Iscove’s modified Dulbecco’s medium, 44% Hams F12, 10% KnockOut Serum Replacement, 1% GlutaMAX-I, 1% Penicillin-Streptomycin (10,000 U/ml), and 20 μM Y-27632. 1.5nM (55 ng/ml) recombinant human BMP4 was added to the culture on day 6, and its concentration was semi-reduced with half medium change every 3 days. Retinal differentiation media Ⅱ [DMEM/F12-Glutamax medium containing 10% Fetal Bovine Serum, 1% N2 supplement, 1% Penicillin-Streptomycin (10,000 U/ml), 0.5 μM retinoic acid and 0.1 mM taurine] was exchanged until day 18 when the aggregate bodies were transferred to 9 cm nonadherent dishes (30 bodies per dish) for further long-term culture in a 37℃, 5% CO2/40% O2 incubator, and half of the volume changed with fresh retinal differentiation media Ⅱ every 5 days. The state and morphology of organoids was monitored in real time using the inverted fluorescence microscope.

Extracted molecule

total RNA

Extraction protocol

Total RNA for each sample was isolated with TRIzol reagent and purified using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions, RNA quality and quantity were assessed using NanoDrop 2000, Agilent 2100 Bioanalyzer and Agilent RNA 6000 Nano Kit.
RNA library construction and RNA sequencing were performed by the annoroad Gene Technology. Sequencing libraries were generated using NEB Next Ultra RNA Library Prep Kit for Illumina (NEB) and library clustering was performed using HiSeq PE Cluster Kit v4-cBot-HS (Illumina) following the manufacturer’s recommendations. After cluster generation, the libraries were sequenced on an Illumina platform and 150 bp paired-end reads were generated.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

RNA sequencing

Data processing

Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to hg38 whole genome.
Reads count and Fragments Per Kilobase of exon per Megabase of library size (FPKM) were calculated.
Genome_build: GRCh38
Supplementary_files_format_and_content: excel files include count and FPKM values for each Sample ...

Submission date

Jul 23, 2021

Last update date

Jul 25, 2021

Contact name

dandan Fan

E-mail(s)

biofandd@gmail.com

Organization name

Wenzhou Medical University

Lab

Institute of Biomedical Big Data