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| Status |
Public on Jan 23, 2022 |
| Title |
NGN2_RNA_seq_line_10 |
| Sample type |
SRA |
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| Source name |
NG-81739
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Glutamatergic, NGN2-induced, day-20 neuron
rutgers university cell and dna repository rucdr-nimh stem cell center index id: 05C45915
disease status: case
gender: Male
age: 52
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| Growth protocol |
Briefly, on Day 0, 5 × 105 hiPSCs were dissociated using Accutase (STEMCELL) and replated in 1.2 ml mTeSR Plus media containing 5 µM ROCK inhibitor, rtTA virus and Ngn2-puro or Ngn2-Hygro virus on 6-well plates. On Day 1, media was refreshed with mTeSRPlus containing 2 µg/ml doxycycline. From days 2 to 4, media was refreshed daily with Neuralbasal Medium supplemented with 1× Glutamax/B27, 2 µg/ml doxycycline, and 2 µg/ml puromycin or 200 µg/ml hygromycin. On day 5, cells were dissociated with Accutase and resuspended in Neuralbasal Medium supplemented with 1× Glutamax/B27, 2 µg/ml doxycycline, and 10ng/ml BDNF/GDNF/NT-3. Cells were replated at 2 × 105/cm2 on 0.1% PEI/Matrigel coated 12-well plates. For morphological immunostaining, 5 × 104 iNs were replated onto 12 mm coverslips (with 5 × 104 rat astrocytes pre-cultured) in Neuralbasal Medium supplemented with 1× Glutamax/B27, 2 µg/ml doxycycline, 10 ng/ml BDNF/GDNF/NT-3, and 1% FBS. From Day 6, media was refreshed every three days with a half volume change. Doxycycline was withdrawn from Day 14 onwards. RNA-seq samples were harvested at Day 30
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| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA-Seq, at the day of harvesting the culture media was removed by aspiration and the cells were washed twice using 1 ml PBS each with aspiration. Subsequently, 600 µl of RLT Plus reagent was directly added into each well to homogenise cells in situ. Qiagen RNEasy plus kit was used for the extraction of total RNA according to the manufacturer’s protocol. Extracted RNAs were eluted into 50 µl of RNA-free water and the concentration was determined by nanodrop. Approximately 5 µg total RNA was recovered from each well.
Total RNAs isolated by using MirVana kit (Thermofisher) were used in RNA-seq. After QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads. First, the mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina), dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Subsequently, terminal repair and sequencing adaptor ligation were applied to the templates. The double-stranded cDNA library is completed through size selection and PCR enrichment.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
NGN2_RNA_seq_line_10 [CD0000010]
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| Data processing |
All raw sequence reads generated by Illumina HiSeq 2000 had been demultiplexed at the University of Minnesota Genomics Center and provided as 2×75 bp paired-end fastq files (targeting 60 M reads per sample).
Adapter remnants, low-quality reads, and low QSEQ short sequences near either end of reads were processed by Trimmomatic (ILLUMINACLIP:NexteraPE-PE.fa:2:30:7, SLIDINGWINDOW:3:18, MINLENGTH:26).
The processed sequences were separated into paired-end and single-end fastq files per sample and only paired-end reads were retained for subsequent mapping. The fastq files were individually mapped against the human genome reference file including decoy sequences (GRCh38p7.13/hg38, 1000 Genome Project) using bowtie2 (-x 2000, -mm --qc-filter –met 1 –sensitive –no-mixed -t) and subsequently merged and sorted as BAM-formatted files using samtools, with only uniquely mapped reads (MAPQ > 30) retained. Picard tools MarkDuplicate was then used to remove all PCR and optical duplicated reads from the BAM file.
To further eliminate allelic bias towards reference alleles during the aligning step, we performed WASP calibration on the generated raw BAM files 55. Briefly, we first called the VCF file profiles on all SNP sites that were not reference alleles of all 20 samples individually using GATK HaplotypeCaller, and subsequently collapsed the individual VCF files into one summary VCF file containing all non-reference sites of all 20 individuals. Subsequently, this SNP list was used as the basis of WASP calibration and re-alignment, and a new WASP-calibrated new BAM file set was collected as the final output for the following peak calling and ASoC SNP call 55.
In order to increase sample size and sensitivity for peak detection, the BAM files of the processed reads of each sample were first sub-sampled to 30 M paired-end reads per sample (the smallest sample size) and the 20 sub-sampled BAM files were merged as the source file for peak calling. MACS2 26 was used to generate peak files (narrowPeak format) with recommended settings at FDR = 0.05 (-f BAMPE, --nomodel, --call-summits --keep-dup-all -B). Peaks that fell within the ENCODE blacklisted regions were removed. Also, we removed peaks falling within chromosomes X and Y, and the mitochondrial genome regions.
Genome_build: GRCh38.p13
Supplementary_files_format_and_content: .narrowPeak, MACS2 peak file
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| Submission date |
Nov 09, 2021 |
| Last update date |
Jan 23, 2022 |
| Contact name |
Jubao Duan |
| E-mail(s) |
jduan69@gmail.com
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| Phone |
(224) 364-7564
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| Organization name |
NorthShore University HealthSystem
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| Department |
Center for Psychiatric Genetics
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| Lab |
Unit of Functional Genomics in Psychiatry
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| Street address |
1001 University Place
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| City |
Evanston |
| State/province |
IL |
| ZIP/Postal code |
60201 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE188491 |
Chromatin accessibility mapping for GWAS risk loci reveals compound genetic effects of neurodevelopment and neurodegenerative disorders in human neurons |
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| Relations |
| BioSample |
SAMN22959286 |
| SRA |
SRX13025734 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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