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| Status |
Public on Apr 15, 2020 |
| Title |
2075_3_XLSB_17_days |
| Sample type |
SRA |
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| Source name |
human iPSC
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| Organism |
Homo sapiens |
| Characteristics |
subjectid: 2075
iPSclinerep: 3
treatmentcondition: XLSB.TesR
daysofneuraldifferentiation: 17
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| Treatment protocol |
The 6 human iPSC lines (2063-1, -2; 2053-2, -6, 2075-1, -3) were differentiated into forebrain NSCs as previously described (Maroof et al., 2013). NSCs were cultured in N2-B27 medium supplemented with XAV939 (2 μM, Stemgent), LDN193189 (100 nM) and SB431542 (10 μM) (XLSB) for 12 days. mTesR medium was gradually switched to N2-B27 in the first 7 days as following: day0 100% mTesR, day1 75%mTesR + 25% N2-B27, day3 50% mTesR and N2-B27, day5 25% mTesR + 75% N2-B27, day7 100% N2-B27. NSCs were passaged on day 8 in N2 + B27 +XLSB; XLSB was withdrawn on day 12. On day 17, NSCs were passaged over astrocytes or without astrocytes and terminally differentiated in Neurobasal medium (NB) + B27 until day 32.
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| Growth protocol |
Monolayer culture of hPSC in feeder-free condition was done following the previously established protocol (Chen et al., 2012). The hPSCs were dissociated to single cells with accutase (A11105, Life Technologies), plated at a density of 1 X 105 cells/cm2 in a Matrigel (354277, BD)-coated plate and cultured with mTeSR1 (05850, Stem Cell Technology). Cells were plated in medium containing 5 μM Y27632, ROCK inhibitor (Y0503, Sigma-Aldrich) to increase cell survival upon dissociation. ROCK inhibitor was removed from the medium at 24 hours after plating and cells were cultured for another 4 days before next passage.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNeasy Mini Kit (Qiagen, Ct# 74106)
RNAseq libraries were constructed using Illumina TruSeq Stranded Total RNA RiboZero Gold sample Prep Kit (RS-122-2303) following the manufacturer’s protocol. The concentration of RNA was measured by Qubit (Life Technologies). Quality of RNAseq library was measured by LabChipGX (Caliper) using HT DNA 1K/12K/HiSens Labchip. The final cDNA libraries were sequenced using HiSeq 3000 for high-throughput DNA sequencing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
R15-364
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| Data processing |
After sequencing run the Illumina Real Time Analysis (RTA) module was used to perform image analysis, base calling, and the BCL Converter (bcl2fastq v2.17.1.14) were followed to generate FASTQ files which contain the sequence reads.
Read-level Q/C was performed by FastQC (v0.11.5). Pair-end reads of cDNA sequences are aligned back to the human genome (UCSC hg19 from Illumina iGenome) by the spliced read mapper TopHat (v2.0.9) with default option with “--mate-innder-dist 160” and “--library-type fr-firststrand” parameters based on known transcripts of Ensembl Build GRCh37.75.
To achieve gene-level expression profile, the properly paired and mapped reads are achieved by “samtools sort –n” option, and these reads are counted by htseq-count v0.9.1 (with intersection-strict mode and stranded option) according to gene annotation (Ensembl Build GRCh37.75) and RPKM is calculated.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include raw counts for each sample
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| Submission date |
Jan 23, 2020 |
| Last update date |
Apr 16, 2020 |
| Contact name |
Carlo Colantuoni |
| E-mail(s) |
ccolantu@jhmi.edu
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| Phone |
4104931439
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| Organization name |
Johns Hopkins Univ. School of Medicine
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| Department |
Neurology
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| Street address |
733 N Broadway
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
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| Platform ID |
GPL21290 |
| Series (2) |
| GSE144157 |
Variation of human neural stem cells generating organizer states in vitro before committing to cortical excitatory or inhibitory neuronal fates [human RNAseq #2] |
| GSE144159 |
Variation of human neural stem cells generating organizer states in vitro before committing to cortical excitatory or inhibitory neuronal fates |
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| Relations |
| BioSample |
SAMN13910543 |
| SRA |
SRX7624766 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4282271_R15-364.count.txt.gz |
95.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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