|
| Status |
Public on Jun 26, 2024 |
| Title |
hESC_FACS_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Regea08/017
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Regea08/017
cell type: induced LSCs
genotype: WT
|
| Treatment protocol |
Induced LSCs were selected via FACS for AREG+, ITGA6+ PODXL-, and cultured an additional 15 days in CNT30 medium.
|
| Growth protocol |
Corneal differentiation of hPSCs was performed with a previously established protocol (Hongisto et al., 2017, 2018). Briefly, hPSCs cultured in Essential 8™ Flex medium (ThermoFisher) on recombinant laminin 521 (L521; Biolamina) were induced towards the surface ectoderm in a 4-day suspension culture, which involved the formation of embryoid bodies with 5 µM Blebbistatin overnight, followed by one day supplementation with bFGF (50 ng/ml) and SB-505124 (10 μM) and two-day supplementation with BMP4 (25 ng/ml). Adherent differentiation from D4 onwards was carried out in CnT-30 medium (CELLnTEC) on Collagen IV (Col IV; Sigma, 5.0 µg/cm2) and L521 (0.5 µg/cm2) combination matrix
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the Qiagen RNeasy® Plus Kit, according to the manufacturer’s protocol. RNA concentrations were measured using the the DeNovix DS-11FX spectrometer.
500 ng of RNA was was prepared for sequencing using the KAPA RNA HyperPrep Kit with. Libraries were sequenced on the Illumina NextSeq 500, generating an average of 15–20 million reads per sample.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
hESC_FACS_rep1
|
| Data processing |
Preprocessing of reads was done automatically with workflow tool seq2science v0.7.1
Paired-end reads were trimmed with fastp v0.20.1 with default options. Genome assembly GRCh38.p13 was downloaded with genomepy 0.11.1. The effective genome size was estimated per sample by khmer v2.0 by calculating the number of unique kmers with k being the average read length per sample. Reads of RNAseq samples were aligned with STAR v2.7.6a with default options. Afterwards, duplicate reads were marked with Picard MarkDuplicates v2.23.8. General alignment statistics were collected by samtools stats v1.14. Mapped reads were removed if they did not have a minimum mapping quality of 30, were a (secondary) multimapper or aligned inside the ENCODE blacklist. RNAseq sample counting and summarizing to gene-level was performed on filtered bam using HTSeq-count v0.12. Sample sequencing strandedness was inferred using RSeQC v4.0.0 order to improve quantification accuracy.
Assembly: GRCh38.p13
Supplementary files format and content: tab-delimited text files include gene count values for each Sample
|
| |
|
| Submission date |
Nov 22, 2023 |
| Last update date |
Jun 26, 2024 |
| Contact name |
Jo Huiqing Zhou |
| E-mail(s) |
jo.zhou@radboudumc.nl
|
| Organization name |
Radboud University
|
| Street address |
Geert Grooteplein 26/28
|
| City |
Nijmegen |
| ZIP/Postal code |
6525GA |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE248495 |
Deciphering the heterogeneity of differentiating hPSC-derived corneal limbal stem cells through single-cell RNA-sequencing [Bulk-seq] |
| GSE248497 |
Deciphering the heterogeneity of differentiating hPSC-derived corneal limbal stem cells through single-cell RNA-sequencing |
|
| Relations |
| BioSample |
SAMN38375271 |
| SRA |
SRX22617906 |
