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| Status |
Public on Feb 20, 2022 |
| Title |
81214-3iPSC RNA-Seq |
| Sample type |
SRA |
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| Source name |
induced pluripotent stem cell
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| Organism |
Homo sapiens |
| Characteristics |
individual: 81214
phenotype: patient with ADCA-DN
cell type: induced pluripotent stem cell
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| Growth protocol |
In order to generate induced neurons (iNs), iPSCs cells were maintained under feeder-free conditions in mTeSR1 media ( STEMCELL Technologies). Media was changed daily. Once cell density reached 75%-80% confluence, clones were dissociated and plated onto 6 well plates. On day 1, cells were infected with lentivirus ( RTTa, GFP and Ngn2) in mTESR1 media. Day 2, media was replace with N2 media and doxycycline. From days 3-6, puromycin selection continued with N2 media changes every day.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Genomic DNA was extracted from fibroblast and iPSC’s using Blood & Cell Culture DNA mini kit (Qiagen) and iN’s using Quick –DNA Miniprep Plus Kit (Zymo Research). Total RNA was extracted from fibroblast, iPSC’s and iN’s using the RNeasy mini kit and RNeasy micro kit (Qiagen).
Libraries were prepared according to KAPA biosystems instructions accompanying KAPA library Preparation Kit Illumina platforms. In brief, genomic DNA was sonicated (Covaris) to generate fragments 180-220 bp in size. Following fragmentation, DNA was used to construct library. After library construction, DNA libraries were bisulfite converted and purified using the EZ DNA Methylation Lightning Kit (Zymo Research). Bisulfite converted samples libraries were amplified using LM-PCR. Amplified bisulfite converted samples and SeqCap Epi probes were hybridized for 64-72 h. Captured DNA samples were washed and recovered, followed by amplification of the capture DNA samples using LM-PCR. Amplified samples were purified using Agencourt AMPure XP Beads (Beckman Coulter). Bisulfite converted libraries captured by SeqCap Epi CpGiant Probes kit sequenced on Illumina HiSeq 4000 platform by 2x150 paired-end. For RNA-seq, library construction was performed using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs) following manufacturer’s instructions. Briefly, total RNA was converted to mRNA using polyA purification. The mRNA was then fragmented and primed for cDNA synthesis. The cDNA was adenylated and adapters were ligated onto the cDNA. Samples were amplified using PCR, amplified library was then purified using Agencount AMPure XP beads (Beckman Coulter). Final libraries were checked for quality on a Bioanalyzer DNA High Sensitivity chip (Agilent). Stand-specific RNA libraries of all the samples were sequenced on Illumina NextSeq 550 platform using 2x150 paired-end sequencing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Data processing |
RNA-Seq data: Cutadapt (Version 1.8.1) was used to trim Illumina TruSeq adapters and low-quality ends from the raw reads. Bowtie2 (Version 2.3.1) was used to align the trimmed reads to the RefSeq transcriptome (GRCh38.p10) and RSEM (Version 1.2.30) was used to quantify gene expression in a strand-specific manner by setting parameter “--forward-prob 0”. DESeq2 (Version 1.12.4) was used to perform differential expression analysis between cell types as well as between patients and controls within each cell type.
Methylation data: Cutadapt (Version 1.8.1) was used to trim Illumina adapters and low-quality ends from the raw reads. The trimmed reads were mapped to human RefSeq genome (GRCh38.p10) using Bismark (Version 0.16.3). Duplicates were removed by the deduplicate_bismark script in Bismark. Only one copy of the overlapping parts in the middle of paired-end reads was retained after clipping the read with the lower average quality in the overlap region by the “clipOverlap” tool in bamUtil (Version 1.0.14). Methylation ratio for each CpG was extracted by the bismark_methylation_extractor script in Bismark. Differentially methylated regions (DMRs) were identified between cell types as well as between patients and controls within each cell type by metilene (Version 0.2-6) with >= 3 CpGs and a mean methylation difference between the two compared groups of >= 0.2. DMRs with FDR corrected p-value < 0.05 were considered significant.
Genome_build: GRCh38.p10
Supplementary_files_format_and_content: RNA-Seq data — gene name, transcript id, length, effective_length, expected count, TPM, FPKM
Supplementary_files_format_and_content: Methylation data — chromosome, CpG position, CpG methylation ratio
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| Submission date |
Feb 21, 2019 |
| Last update date |
Feb 20, 2022 |
| Contact name |
Xianglong Zhang |
| Organization name |
Amgen Inc.
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| Department |
Department of Cardiometabolic Disorders
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| Street address |
One Amgen Center Dr.
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| City |
Thousand Oaks |
| State/province |
CA |
| ZIP/Postal code |
91320 |
| Country |
USA |
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| Platform ID |
GPL21697 |
| Series (1) |
| GSE126890 |
Tissue Specific Methylation and Expression Patterns of ADCA-DN Patients |
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| Relations |
| BioSample |
SAMN10984963 |
| SRA |
SRX5405768 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3617768_81214-3iPSC.genes.results.txt.gz |
643.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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