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Sample GSM6351540

Query DataSets for GSM6351540

Status

Public on Jun 23, 2024

Title

Control 2 [IonCode_0123]

Sample type

SRA

Source name

iPSC

Organism

Homo sapiens

Characteristics

group: Control
cell line: iPSC
cell type: Neuron

Treatment protocol

no treatments were applied

Growth protocol

All five iPSC lines were cultured and differentiated in parallel, with a minimum of three independent differentiation batches for each experimental assay. NGN2-induced cortical neurons were differentiated according to the protocol published by Zhang et al. in 2013 [13], with modifications described below. On differentiation day -2, iPSCs were dissociated with Accutase (Innovative Cell Technologies #AT104) and plated at 90,000 cells/cm2 in mTeSR-1 media supplemented with 10uM ROCK inhibitor (Y-27631, Cayman Chemical #10005583), on Geltrex-coated 12-well plates. The following day, on differentiation day -1, iPSCs were transduced with lentiviral vectors expressing NGN2, eGFP, and rtTA along with 8ug/ml polybrene (Sigma-Aldrich #TR-1003-G). High quality lentiviral particles were produced and concentrated at the Viral Core at Boston Children’s Hospital. Plasmids used were gifts from the Thomas Sudhof laboratory, but all components can be purchased from Addgene with the following IDs: 52047, 30130, 20342. On differentiation day 0, NGN2 and eGFP expression was induced via treatment with 2ug/ml doxycycline (Millipore #324385). Cells that were positive for infection with the viral vectors were selected via treatment with 1ug/ml puromycin (Invitrogen #ant-pr-1) for 24-48 hours, depending on cell density. This selection helps to generate a relatively pure population of neurons. For the first 2 days, the following growth factors and supplements were added to the N2 medium: 10ng/ml BDNF (Peprotech #450-02), 10ng/ml NT3 (Peprotech #450-03), and 0.2mg/L laminin (ThermoFisher #23017-015). Differentiating cells were then fed with B27 media containing the following growth factors and supplements every other day until differentiation day 6: 10ng/ml BDNF, 10ng/ml NT3, 0.2mg/L laminin, 2ug/ml doxycycline, and 2uM Ara-C (Sigma-Aldrich #C1768). Contrary to the originally published protocol, on day 2, no mouse glial cells were added. Instead, astrocyte-conditioned media was collected from cultures of iPSC-derived astrocytes (NCardia Astro.4U) and neurons were cultured in this human-astrocyte-conditioned media along with growth factors and supplements after day 6 to avoid astrocyte signals in the RNA analysis. Neurons were matured until day 14 prior to being collected for RNA analysis.

Extracted molecule

total RNA

Extraction protocol

At day 14, neurons were collected by removing media and adding a thin layer of lysis buffer from the RNA purification kit and using a cell scraper to collect the cells off the plate. Total RNA was isolated using Clontech RNA isolation kit #740971.50
Isolated total RNA was diluted to 0.95ng/ul and 10.5ul was loaded in each reverse transcription reaction with the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher #11754050). Reverse transcription was completed in a thermocycler with the following temperature settings: 42°C 30minutes, 85°C for 5 minutes, 10°C Hold. After reverse transcription, libraries were prepped using the Ion Chef (ThermoFisher Cat#4484177) and the Ion AmpliSeq Transcriptome Human Gene Expression Panel, Chef-Ready Kit (ThermoFisher Cat#A31446). The AmpliSeq Gene Expression Panel includes one primer pair for almost every gene in the transcriptome, allowing for amplification of a single 100bp amplicon for each gene to be sequenced. Libraries from 8 samples were pooled together, templated, and loaded on the Ion 540 Chip (ThermoFisher Cat#A27765) using the Ion Chef and the Ion 540 Kit Chef (ThermoFisher Cat#A27759). After templated samples were loaded onto the 540 Chips, each Chip was inserted into the Ion Torrent S5 sequencer (ThermoFisher Cat#A27212) and 500 flows were applied for each sequencing run. The second Chip was stored at 4°C during the first sequencing run and removed to room temperature for 30 minutes prior to running the second Chip.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Ion Torrent S5

Data processing

Read mapping and preliminary data analysis was completed with the AmpliSeq Plugin available on the ThermoFisher S5 Torrent server. CHP files were loaded into the Transcriptome Analysis Console (TAC) Software from ThermoFisher to complete differential gene expression analysis of the probands versus controls, with 1.2-fold change in expression as cutoff and p-values corrected for multiple comparisons using FDR. Gene ontology analysis was completed using DAVID (https://david.ncifcrf.gov).

Submission date

Jul 19, 2022

Last update date

Jun 23, 2024

Contact name

Kellen Winden

Organization name

Boston Children's Hospital

Department

Neurology

Street address

300 Longwood Ave