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| Status |
Public on Oct 13, 2022 |
| Title |
HS360-Day13-rep1 [10-sc1-D13-ctrl-A] |
| Sample type |
SRA |
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| Source name |
HS360, day 13, past stage II self-patterning
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| Organism |
Homo sapiens |
| Characteristics |
source cell line/type: Human embryonic stem cells HS360
time point: Day 13
gender: male
tissue/cell type: self-patterning NPCs
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| Treatment protocol |
In brief, at Day 0 HS360 hESCs grown in E8 were collected with Accutase and seeded in Geltrex precoated 12 well dishes. Single-cell suspensions were transferred to E8 medium containing 10 µM Rock inhibitor and at Day 1, the culture medium was switched to neural induction medium (NIM; Advanced DMEM/F12, 1% GlutaMAX, 1% Penicillin/Streptomycin, 1% N2). During days 2 to 6, media were changed daily and the LSX combo (LDN-193189, SB431542, XAV939) was added fresh to NIM. Cells were collected at Day 7 for passaging into the self patterning phase, counted and seeded on 12 well plates sequentially precoated with polyornithine (PO), fibronectin (F) and Geltrex. At Day 7 the Stage II neural self-patterning (NSPM) medium (Advanced DMEM/F12, 1% GlutaMAX, 1% Penicillin/Streptomycin, 1% N2, 1% B27) was supplemented with 10 µM Rock inhibitor. Culture medium was changed daily till day 13. At Day 13, cells were collected for passaging into the maturation phase, counted and seeded on precoated PO+F+Geltrex 12 well dishes and Stage III Neural maturation medium (NMM; Advanced DMEM/F12, 1% GlutaMAX, 1% Penicillin/Streptomycin, 1% N2, 0.5% B27, 10 ng/ml FGF2, 10 ng/ml EGF) was changed daily till Day 20, where cells were collected.
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| Growth protocol |
Human embryonic stem cells HS360 (Karolinska Institutet, Sweden, RRID:CVCL_C202) were maintained in Essential 8™ Medium, in feeder-free conditions on Geltrex™ Matrix solution pre-coated culture plates.Culture media were replaced daily and cells were routinely passaged at 75-85% confluency using 0.5 mM EDTA in ratios between 1:3 to 1:6.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cultured cells using Norgen RNA/DNA purification kit or Qiagen RNeasy kit and treated with DNase according to manufacturer´s instructions.
Illumina TruSeq Stranded mRNA Library Prep kit was used to contruct the sequencing library according to manufacturer´s instructions. Libraries were sequenced on an Illumina Novaseq 6000 S1 flow cell with 100bp pair end reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
BBDuk (BBMap v. 34.56) was used to reomve and trim low-quality reads and adaptor sequences with the following configurations: ktrim=r k=23 mink=11 hdist=1 tbo tpe qtrim=r trimq=15 maq=15 minlen=36 forcetrimright=149
Reads were mapped to the human genome/transcriptome ENSEMBL GRCh38.p5 release 83 using hisat2 v2.1.0
FeatureCounts v.1.4.6-p1 was used to count the reads mapping to the features using default configurations
Downstream normalisation and differential expression analysis was performed using R software DEseq2 package v 1.30.1.
Genome_build: ENSEMBL GRCh38.p5 release 83 (genome-build-accession NCBI:GCA_000001405.20)
Supplementary_files_format_and_content: Count files with data matrix with unnormalized counts for each sample. Additionaly, "normcounts.control.csv" contains a normalized count matrix for all samples.
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| Submission date |
Dec 31, 2021 |
| Last update date |
Oct 15, 2022 |
| Contact name |
Ragnhild Eskeland |
| E-mail(s) |
ragnhild.eskeland@medisin.uio.no
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| Phone |
(+47) 22 85 14 57
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| Organization name |
University of Oslo
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| Department |
Institute of Basic Medical Sciences
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| Lab |
Chromatin Biology
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| Street address |
Sognsvannsveien 9
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| City |
Oslo |
| ZIP/Postal code |
0371 |
| Country |
Norway |
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| Platform ID |
GPL27644 |
| Series (2) |
| GSE192855 |
Gene expression analysis of hESCs undergoing neuronal differentiation [RNA-seq] |
| GSE192858 |
Epigenome analysis and Gene expression analysis of hESCs undergoing neuronal differentiation |
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| Relations |
| BioSample |
SAMN24547313 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5766884_10-sc1-D13-ctrl-A.counts.txt.gz |
2.5 Mb |
(ftp)(http) |
TXT |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
| Raw data not provided for this record |
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