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| Status |
Public on Aug 05, 2020 |
| Title |
NEUR_D3_rep3 |
| Sample type |
SRA |
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| Source name |
Developing Neuron
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| Organism |
Homo sapiens |
| Characteristics |
sample group: NEUR_D3
differentiation days: 3
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| Treatment protocol |
NA
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| Growth protocol |
For the three human iPSC lines, one was derived from fibroblasts and two from peripheral blood mononuclear cells. All three of these lines were then reprogrammed via episomal plasmids and grown in Essential 8 medium of a 6-well plate with daily medium changes (Chen et al, 2012). The Neural stem cells (NSCs) were derived from three episomally reprogrammed iPSC lines and one human ES cell line. They were expanded in a neurobasal medium containing B27 and FGF2 (Efthymiou et al., 2014). Where after, neuronal differentiation was initiated by medium exchange. The Mature Neuron sample was obtained by growing NSCs for 21 days in neuronal differentiation medium (Shaltouki et al., 2013).
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extractions of iPSCs and NSCs were performed when cells were 70% confluent and at the indicated time point for the differentiated Neurons and Astrocytes. All RNA was extracted with the RNAesy Mini Kit (Qiagen).
Libraries for each RNA extracted were prepared using 10ug in conjunction with the Illumina single-end Total RNA-Seq ribosome depletion kit. Post preparation, libraries were single-end seqeunced (50bp) for >30M reads each.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Developing Neuron Day 3 replicate 3
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| Data processing |
RTA 1.13.48 and CASAVA 1.8.2. software used for basecalling and deplexing.
Post CASAVA deplexing, .fastq files generated per library were import into the CLCbio Genomics Workbench (www.clcbio.com) and QC inspected, adaptor clipped, low quality trimmed, filtered, then mapped. Per quality trimming, 15bp from the 5' end of each read was globally removed along with 1bp from the 3' end. Bases with a call accuracy < 95% were trim-removed thereafter. Per filtering, reads having more than two ambiguities were discarded along with those having read length <15bp post post quality trimming. Per mapping, the "RNA-Seq" tool supported in the Workbench was used to locally align reads by library against the human genome (GRCh38/hg38) without masking; keeping only those mapped reads with >= 80% reference identity for >=80% read length. Post mapping, the number of reads falling in a RepeatMasker annotated "HERV" region (Library 20120124) were enumerated and the counts converted into "Reads Per Kilo base of transcript per Million mapped reads" ("RPKM") expression values. RPKM expression values were next organized by HERV region across libraries and imported into R (https://cran.r-project.org/). In R, RPKM expression values were pedestalled by 2, Log2 transformed, filtered to remove regions not having at least one transformed value >1, then quantile normalized. Normalized values for remaining regions were then used to assess for and remove library-level outliers via tukey box plot, covariance-based PCA scatter plot, and pearson correlation heat map. For libraries not removed as outliers, normalization of RPKM expression values was repeated and the new normalized expression noise modeled using LOWESS (CV~mean). Based on the LOWESS fit, regions not having at least one normalized value >=2.00 were considered noise-biased and subsequently discarded as such. For regions not discarded, differential expression across libraries was tested for using the one-factor ANOVA test under BH FDR MCC condition using sample type as the factor. Regions having a corrected P < 0.05 by this test were subset and deemed to be those having differential expression across the sample types tested. For those regions subset only, the TukeyHSD post-hoc test was further applied to identify which regions between sample types have an uncorrected P <0.05 and an absolute difference of means >=2. These regions were subsequently deemed to be differentially expressed between the two sample types compared.
Genome_build: hg38
Supplementary_files_format_and_content: RPKM enumerated expression for each RepeatMasker annotated HERV region in hg38. Also provided are .txt files that describe the cross sample RPKM expression before normalization, after normalization (pre-noise filtering), and after noise filtering.
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| Submission date |
Feb 07, 2018 |
| Last update date |
Aug 05, 2020 |
| Contact name |
Kory R Johnson |
| E-mail(s) |
johnsonko@ninds.nih.gov
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| Phone |
301-402-1956
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| Organization name |
NINDS/NIH
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| Department |
DIR IT & Bioinformatics
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| Lab |
Bioinformatics Section
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| Street address |
10/3B01, 9000 Rockville Pike
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL9115 |
| Series (2) |
| GSE110267 |
Human Endogenous Retrovirus-K in Stem Cell Function and Neuronal Differentiation [dataset 1] |
| GSE110498 |
Human Endogenous Retrovirus-K in Stem Cell Function and Neuronal Differentiation |
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| Relations |
| BioSample |
SAMN08475024 |
| SRA |
SRX3656597 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2983775_RPKM_Expression_Enumeration_NEUR_D3_rep3.txt.gz |
11.1 Mb |
(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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