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Sample GSM3672571

Query DataSets for GSM3672571

Status

Public on Oct 30, 2020

Title

8-2607-C

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: Wild type
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 At Day 17, Ara-C was completely withdrawn by full medium change, followed by half medium changes until the neurons were harvested around day 21-24.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