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| Status |
Public on Jan 31, 2023 |
| Title |
Neuron-L1C2 Day21 Cocaine Treated |
| Sample type |
SRA |
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| Source name |
iPSC-Neuron
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| Organism |
Homo sapiens |
| Characteristics |
uthbc subject_id: UTHBC0090
tissue: iPSC-Neuron
cell line: Neuron-L1C2
cell type: iPSC-Neuron
treatment: 10 micromolar cocaine, 7 Days
ethnicity: Black
Sex: Male
age at_time_of_death: 58
postmortem interval_(hours): 29.7
rna integrity_number: 8.4
brain cerebellar_ph: NA
postmortem consenus_diagnosis: Cocaine Use Disorder
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| Treatment protocol |
10mM morphine stocks were prepared by dissolving morphine sulfate (Sigma-Aldrich, M8777) in PBS. 10mM cocaine stocks were prepared by dissolving cocaine hydrochloride (Sigma-Aldrich C5776) in PBS. Stocks of cocaine and morphine were stored at 4C until use at which time either morphine or cocaine was diluted in NDM to produce a final concentration of 10µM. To model chronic drug exposure in OUD and CUD, Neuron lines derived from two iPSC clones from one individual with OUD who died of an opioid overdose (morphine and hydrocodone, Table 1, Subject 2) were treated with 10µM morphine or left untreated for 7 days during differentiation from day 14 to day 21 of the neuron differentiated protocol. Morphine-containing or morphine-free media was changed daily for 7 days. Neuron lines derived from two iPSC clones from one individual with CUD who died of a cocaine overdose (Table 1, Subject 1) were treated with 10µM cocaine or left untreated for 7 days during differentiation from day 14 to day 21 of the neuron differentiated protocol. Cocaine-containing or cocaine-free media was changed daily for 7 days. For each cell line, treated and untreated cells were pelleted at day 21 of differentiation for RNA seq analysis.
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| Growth protocol |
At autopsy, three 3 millimeter (mm) skin punches were collected from the forearm of Subject 1 and Subject 2 and dermal fibroblasts were cultured and expanded. Briefly, punches were dissected into 0.5 mm pieces in phosphate buffered saline (PBS) without calcium and magnesium (Corning, Corning, NY) with 100 micrograms (µg)/ milliliter (mL) primocin (InvivoGen, San Diego, CA) and 1x Pen/Strep (Gibco-Thermo Fisher Scientific, Waltham, MA), incubated in 2units/mL type 2 collagenase (Worthington Biochemical, Lakewood, NJ) in Dulbecco’s Modified Eagle Media (DMEM, Corning) overnight, rinsed in DMEM and plated on 3 wells of a of a 24-well culture plate coated with Bovine plasma fibronectin (EMD Millipore, Darmstadt, Germany). Cells were cultured in fibroblast media consisting of 10% FBS (Gibco) and 1x PenStrep (Gibco) in DMEM and passaged and expanded using 0.05% Trypsin/0.53 millimolar (mM) EDTA (Corning) chemical dissociation. Cells were expanded into T-125 flasks and frozen to -80°C in a Mr. FrostyTM freezing container (ThermoFisher) at passage 3 in 1mL of 10% dimethylsufoxide (DMSO, Sigma-Aldrich, St. Louis, MO) at 1.5 million cells per cryovial. Cells were transferred after 24 hours (hr) to long term storage in liquid nitrogen until use when they were thawed quickly in a 37°C water bath, immediately diluted in fibroblast media, and centrifuged before plating to remove DMSO.
Postmortem fibroblasts were induced to iPSCs using the Cyto-TuneTM-iPS 2.0 Sendai Reprogramming Kit (Invitrogen-Thermo Fisher Scientific, Waltham, MA), following manufacturer’s protocol. Briefly, fibroblasts were thawed and plated at passage 4 on 3 wells of a 6-well plates to 50% confluency and then were transfected overnight with the Cyto-TuneTM-iPS 2.0 Sendai viral vectors in fibroblast media at the following Multiplicity of Infection (MOI): KOS = 5, hc-Myc = 5, hKlf4 = 3. After 6 days, cells were replated on 60mm dishes coated with GeltrexTM LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Gibco). Cells were maintained in TeSRTM-E8TM media (StemCell Technologies, Vancouver, BC, Canada) and media was changed daily until iPSC colony formation (10-28 days). Colony clones were manually selected based on morphology and subsequently expanded in mTeSRTM Plus media (StemCell Technologies). Colonies were passaged for expansion using 10 micromolar (µM) EDTA in PBS without calcium and magnesium and gentle mechanical dissociation with a P1000 pipette. 2 clones from each line were selected based on morphology and iPSC marker positivity for expansion and differentiation into neural cells.
Neural progenitor cells (NPC) were generated using StemCell Technologies AggrewellTM plates to generate embryoid bodies (EBs) and then cultured using the StemDiffTM SMADi Neural Induction Kit, following manufacturer’s protocol as previously described (Stertz et al., 2021). After 7 days, EBs were plated in Geltrex-coated 6-well plates in STEMDiff Neural Induction Medium + SMADi, and rosettes were manually selected 7 days later and replated on Geltrex-coated wells for expansion. NPCs were passaged at 80-90% confluence using Accutase cell detachment solution (Innovative Cell Tech, San Diego, CA) and expanded in Neural Basal (NB) media (50% DMEM/F12 (Corning), 50% Neurobasal Medium (Gibco), 1x GlutaMAX (Gibco), 1x NEAA (Gibco), 1x PenStrep (Gibco), 1x N-2 (Gibco), 1x B27 minus vitamin A (Gibco) supplemented with 20ng/mL of recombinant human FGF-Basic (1-155 a.a, PeproTech, Cranbury, NJ).
Cortical neurons were differentiated as previously described (Stertz et al., 2021). NPCs were plated on Laminin/Poly-L-Ornithine coated 6-well plates at a density of 20,000 cells/cm2 and media was replaced the next day with Neuron Differentiation (ND) media consisting of NB media without FGF-Basic supplemented with 20 nanogram (ng)/ml BDNF (PeproTech), 20 ng/ml GDNF (PeproTech), 1 mM dibutyryl-cyclic AMP (Sigma-Aldrich), 200 nanomolar (nM) ascorbic acid (Sigma-Aldrich), 10 ng/mL IGF-1 (PeproTech), and 10 ng/ml WNT-3A (R&D Systems, Minneapolis, MN). ND media was changed every other day until the final neuron time points of Day 7, Day 14, or Day 21.
To avoid cell passage number affecting epigenetic age and cellular maturity, all cell samples were collected, induced and differentiated at the same passage number: passage 4 for fibroblasts, passage 16 for iPSCs, and passage 4 for NPCs.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lifted in Trypsin, Accutase or 10 µM EDTA in PBS without calcium and magnesium, pelleted in 1.5mL tubes and stored at -80oC until use. Fibroblasts were pelleted at 1.5 million cells, iPSCs were pelleted from 2 wells of a 6 well plate at 60-80% confluence, NPCs were pelleted from 2 wells of a 6 well plate at 100% confluence, and neurons were pelleted from 2 wells of each 6 well differentiation plate. RNA was collected from dry frozen pellets using the RNeasy Plus Mini kits (Qiagen, Hilden, Germany).
400ng RNA per sample was used for mRNA library construction using NEBNext® Ultra™ RNA Library Prep Kit (Illumina Inc, San Diego, CA). Paired-end sequencing reads (150bp) were generated on an Illumina HiSeq2000 platform (Novogene Bioinformatics Institute, Chula Vista, CA).
For cell samples: Cells were lifted in Trypsin, Accutase or 10 µM EDTA in PBS without calcium and magnesium, pelleted in 1.5mL tubes and stored at -80oC until use. Fibroblasts were pelleted at 1.5 million cells, iPSCs were pelleted from 2 wells of a 6 well plate at 60-80% confluence, NPCs were pelleted from 2 wells of a 6 well plate at 100% confluence, and neurons were pelleted from 2 wells of each 6 well differentiation plate.
For cell samples: RNA was collected from dry frozen pellets using the RNeasy Plus Mini kits (Qiagen, Hilden, Germany).
For Brain Tissue: RNA was extracted from 50mg of BA9 tissue using RNeasy Plus Mini kits (Qiagen, Hilden, Germany) and RNA integrity number (RIN) was measured for RNA quality (Agilent Bioanalyzer 2100 system, Agilent Technologies, Santa Clara, CA).
For Cells: 400ng RNA per sample was used for mRNA library construction using NEBNext® Ultra™ RNA Library Prep Kit (Illumina Inc, San Diego, CA). Paired-end sequencing reads (150bp) were generated on an Illumina HiSeq2000 platform (Novogene Bioinformatics Institute, Chula Vista, CA).
For Brain Tissue: 1 μg RNA per sample was used for mRNA library construction using NEBNext® Ultra™ RNA Library Prep Kit (Illumina Inc, San Diego, CA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Neuron-L1C2 Day21 Treated
counts-cells-GEO_1-26-23.csv
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| Data processing |
Reads were mapped to the GRCh38 genome using STAR version 1.7.10a. (Dobin et al., 2013)
Gene and transcript counts were obtained using featureCounts (Liao et al., 2014), and from the raw counts, gene lengths, library size, and counts per million were calculated.
Genes with CPM > 1 in at least 1 sample were kept for analysis.
Assembly: GRCh38
Supplementary files format and content: BA9_CUD_GEO-counts.csv: Raw counts matrix of postmortem cocaine use disorder Brodmann Area 9 samples from the UTHealth Brain collection. Columns refer to individual UTH Brain Collection ID for each subject
Supplementary files format and content: counts-cells-GEO_1-26-23.csv: Raw counts matrix of postmortem-derived fibroblast, iPSC, NPC, and Neuron lines from the UTHealth Brain Collection. Columns refer to individual cell line samples and whether they were treated or untreated with corresponding drug (morphine or cocaine)
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| Submission date |
Jan 31, 2023 |
| Last update date |
Jan 31, 2023 |
| Contact name |
Consuelo Walss-Bass |
| E-mail(s) |
[email protected]
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| Organization name |
University of Texas Health Science Center at Houston
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| Department |
Louis A. Faillace Department of Psychiatry and Behavioral Sciences
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| Lab |
Laboratory of Consuelo Walss-Bass PhD
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| Street address |
1941 East Road Rm. 3110
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| City |
Houston |
| State/province |
Texas |
| ZIP/Postal code |
77047 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE224096 |
RNA sequencing of postmortem-fibroblast derived cell lines and postmortem human Brodmann Area 9 from Cocaine Use Disorder Subjects in the University of Texas Health Science Center at Houston Brain Collection |
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| Relations |
| BioSample |
SAMN32971541 |
| SRA |
SRX19226783 |
| 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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