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Sample GSM3672558 Query DataSets for GSM3672558
Status Public on Oct 30, 2020
Title 15-2607-i1
Sample type SRA
Source name National Institute of Mental Health (NIMH) childhood-onset schizophrenia (COS) cohort
Organism Homo sapiens
Characteristics cell type: hiPSC-derived NGN2-neurons
genotype/variation: ATP6V1A knock down
treatment: none
Treatment protocol Human β-amyloid (1-42) peptide was purchased from GenScript (#RP10017). 500 µM β-amyloid was prepared in the vehicle solution of Tris-HCl, pH 7.5 and stored at –20°C. 21-day isogenic pairs of ATP6V1A-manipulated NGN2-neurons were exposed to 5 µM β-amyloid for 24 hours, and then collected for RNA sequencing.
Growth protocol NPCs were seeded as 4-6x10e5 cells/well in a 24-well plate coated with Matrigel (coverslips are put in a plate and coated with Matrigel for immunostaining). At day -1, cells were transduced with rtTA, pLV-TetO-hNGN2-eGFP-Neo and ATP6V1Ai gRNA or empty lentiguide-Hygro-mTagBFP2 (Addgene 99374) lentiviruses via spinfection. Medium was switched to non-viral medium three hours post-spinfection. At Day 0, 1 μg/ml dox was added to induce NGN2-expression. At Day 1, transduced hiPSC-NPCs were treated with corresponding antibiotics to the lentiviruses (300 ng/ml puromycin for dCas9-effectors-Puro, 1 mg/ml G-418 for hNGN2-eGFP-neo and 1 mg/ml HygroB for lentiguide-Hygro-mTagBFP2) in order to increase the purity of transduced NPCs. At day 3, NPC medium was switched to neuronal medium (Brainphys (Stemcell Technologies, #05790), 1x N2 (Life Technologies #17502-048), 1x B27-RA (Life Technologies #12587-010), 1 μg/ml Natural Mouse Laminin (Life Technologies), 20 ng/ml BDNF (Peprotech #450-02), 20 ng/ml GDNF (Peptrotech #450-10), 500 μg/ml Dibutyryl cyclic-AMP (Sigma #D0627), 200 nM L-ascorbic acid (Sigma #A0278)) including 1 μg/ml Dox, along with antibiotic withdrawal. 50% of the medium was replaced with fresh neuronal medium (lacking dox once every second day. At day 11, full medium change withdrew residual dox completely. At day 13, NGN2-neurons were treated with 200 nM Ara-C to reduce the proliferation of non-neuronal cells in the culture, followed by half medium change by day 17. At Day 17, Ara-C was completely withdrawn by full medium change, followed by half medium changes until the neurons were harvested around day 21-24.
Extracted molecule total RNA
Extraction protocol Add 0.25 mL of TRIzol™ Reagent (Thermo Fisher Scientific, Cat#15596026) per 24-well well directly to the culture dish to lyse the cells. Transfer the lysate to 1.5 mL RNase-free eppendorf tube. Add 50 µL of chloroform in the hood, and then securely cap the tube. Invert 2-3 times and vortex 5-10 sec. Hold on ice for 10 min, inverting every 3 min. Centrifuge the sample for 15 minutes at 12,000× g at 4°C. Transfer clear supernatant to fresh RNase-free tubes with 150 µL of Isopropanol. Centrifuge the sample for 10 minutes at 12,000× g at 4°C. Discard the supernatant with a micropipettor. The pellet is washed in 0.5 mL of ice cold 70% ethanol from -20°C. Resuspend the pellet in 50 μL of RNase-free water. Store the RNA at -70°C.
RNA sequencing libraries were prepared using the Kapa Total RNA library prep kit.
NovaSeq, 100nt, Paired-End Read
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
Description processed data file: expression.log2cpm.tsv
Data processing Paired-end sequencing reads (100bp) were generated on a NovaSeq platform. Raw reads were aligned to hg19 using STAR aligner (v2.5.2a).
Following read alignment, featureCounts (1.6.3) was used to quantify the gene expression at the gene level based on Ensembl gene model GRCh37.70.
Genes with at least 1 count per million (CPM) in at least 1 sample were considered expressed and hence retained for further analysis, otherwise removed.
After filtering, the raw read counts were normalized by the voom function in limma.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: expression.log2cpm.tsv: Tab-separated text file.
Submission date Mar 15, 2019
Last update date Oct 30, 2020
Contact name Bin Zhang
Organization name Icahn School of Medicine at Mount Sinai
Department Genetics and Genomic Sciences
Street address 1470 Madison Avenue
City New York
State/province NY
ZIP/Postal code 10029
Country USA
Platform ID GPL24676
Series (1)
GSE128367 Transcriptomic analysis of ATP6V1A knock-down and amyloid beta-treated neurons
BioSample SAMN11134275
SRA SRX5527217

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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