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| Status |
Public on Jul 06, 2023 |
| Title |
AA pre AA H1 R1 |
| Sample type |
SRA |
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| Source name |
H1
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| Organism |
Homo sapiens |
| Characteristics |
cell line: H1
cell type: differentiated human embryonic stem cell
treatment: Ascorbic Acid 100 microM 3 weeks, Ascorbic Acid 100 microM 1h, RNA Extraction
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| Treatment protocol |
To perform tonic neurotransmitter preincubations hMSN-like and interneuron-like cells were grown to DIV45, half-media was removed, and new media containing either ascorbic acid (100 µM in water; Sigma, St. Louis, MO, USA), dopamine hydrochloride (10 nM in 100 µM ascorbic acid; Sigma, St. Louis, MO, USA), acetylcholine chloride (750 nM in water; Sigma, St. Louis, MO, USA), or L-glutamic acid (2 µM in water; Sigma, St. Louis, MO, USA) was added and left in the wells to be incubated at 37 °C until the next day. This process was repeated every day for 21 days, then RNA extracted for sequencing. To perform antagonist incubations, hMSN-like cells were grown to DIV45, half-media was removed, replaced with media containing both R(+)-SCH23390 hydrochloride (250 µM; Sigma, St. Louis, MO, USA), S(-)-Sulpiride (250 µM; Sigma, St. Louis, MO, USA) was added and incubated at 37 °C for 60 min. Half media was then removed and media containing either dopamine hydrochloride (1 mM in 100 µM ascorbic acid; Sigma, St. Louis, MO, USA), R(+)-SCH23390 hydrochloride (250 µM; Sigma, St. Louis, MO, USA), S(-)-Sulpiride (250 µM; Sigma, St. Louis, MO, USA), or PBS (1X; Gibco), individually or in combination, was added and incubated at 37 °C for 60 min then extracted RNA for sequencing. Chronic incubation proceeded by removing all culture media, replacing with maturation media, and repeating dopamine, SCH23390, and Sulpiride incubations for 5 days. Chronic time-course doses were time-matched to extract all samples on DIV50. To perform acute incubations, hMSN-like cells were grown to DIV45, half-media was removed, replaced with dopamine hydrochloride (10 nM–1 mM in 100 µM ascorbic acid; Sigma, St. Louis, MO, USA), L-glutamic acid (5 µM in water; Sigma, St. Louis, MO, USA), SKF82958 (1–100 µM; Sigma, St. Louis, MO, USA), quinpirole (1–100 µM; Sigma, St. Louis, MO, USA), or CGS21680 (0.1–10 µM; Sigma, St. Louis, MO, USA), and incubated at 37 C for 60 min prior to harvesting for RNA-seq. Withdrawal incubations were performed by dosing as previously described for chronic dopamine for 5 days, then ceasing dopamine dosage for two weeks. A final dopamine dosage was performed after 2 weeks and RNA extracted. All doses were performed at 37 C for 60 min.
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| Growth protocol |
hMSN-like cells were obtained following previously described protocols [15] and have been described in detail in prior work [51]. Briefly, the feeder-independent H9 and H1 hESCs (WA09 and WA01; WiCell, Madison, WI, USA) were maintained in 6-well tissue culture dishes (Greiner Bio-One, Alphen aan den Rijn, NL) coated with 0.5 µg/mL reduced growth factor Matrigel solution (Corning, Durham, NC, USA) in E8 medium (Stemcell Technologies, Cambridge, MA, USA) and passaged using standard protocols. Cells were grown to ~75–80% confluency prior to differentiation. On days in vitro (DIV) 0 of differentiation, E8 medium was removed, cells were washed with 1X PBS (Gibco, Waltham, MA, USA), and media switched to DMEM-F12/Neurobasal media (2:1) (Gibco) supplemented with N2 (Gibco) and B27 minus Vitamin A (Gibco) (together referred to as N2B27). From DIV 0 to 4, cultures were supplemented with SB431542 (10 µM in 95% EtOH; Selleck Chemicals, Houston, TX, USA), LDN-193189 (100 nM in DMSO; BioVision, Waltham, MA, USA), and dorsomorphin (200 nM; BioVision). Media was changed every day. From DIV 5 to 8, cultures were supplemented with just LDN-193189 and dorsomorphin. Media was changed every day. On DIV 9, cultures were washed with 1X PBS, and media switched to N2B27 supplemented with activin A (25 ng/mL in 4 mM HCl; R&D, Minneapolis, MN, USA). Cells were then lifted using cell scrapers (Greiner Bio-One) since EDTA treatment drastically reduced survivability, pipetted up and down 1 time with a 5 mL serological pipette, and replated with 5.21E5 cells/cm2 onto Matrigel-coated 6-well plates. Half-media was changed the next day and then every other day afterward. On DIV 18, cultures were passaged using 0.5 mM EDTA for 2 min at 37 °C, as we no longer observed issues with survivability with EDTA treatment, and pipetted up and down 10–15 times using P1000 mechanical pipette then replated onto Poly-L-Ornithine (15 µg/mL in water; Sigma, St. Louis, MO, USA) and Laminin (5 µg/mL in PBS; Corning) coated 24-well plates (Greiner Bio-One) at 2.11E5 cells/cm2. Half-media was changed the next day and then every other day afterward. From DIV 20 to 24, media was switched to N2B27 with Vitamin A (Gibco). On day 25 for analysis, BDNF and GDNF (10 ng/mL in 0.1% BSA (w/v); Peprotech) were added to aid neuronal maturation and survival (referred to as Maturation Medium). Cells were lifted, similar to the DIV18 passage, on DIV 30 to 35 and all cells were replated onto Poly-L-Ornithine (2 µg/cm2) and Laminin (1 µg/cm2) coated 24-well plates to maintain neuron attachment. Cells were maintained in a humid incubator at 37 °C with 5% CO2. Striatal interneuron-like cells were obtained following a previously published differentiation protocol [55]. H9 and H1 hESCs were grown to ~80-90% confluency prior to differentiation. On days in vitro (DIV) 0 of differentiation, E8 medium was removed, cells were washed with 1X PBS (Gibco, Waltham, MA, USA), and media switched to DMEM-F12/Neurobasal media (2:1) (Gibco) supplemented with N2 (Gibco) and B27 (Gibco) (together referred to as N2B27). From DIV 0 to 9, cultures were supplemented with SB431542 (10 µM in 95% EtOH; Selleck Chemicals, Houston, TX, USA), LDN-193189 (100 nM in DMSO; BioVision, Waltham, MA, USA), and XAV939 (2 µM in DMSO; R&D, Minneapolis, MN, USA). Media was changed every day. On DIV 9, cells were lifted using cell scrapers (Greiner Bio-One) since EDTA treatment drastically reduced survivability, pipetted up and down 1 time with a 5 mL serological pipette, and replated with 5.21E5 cells/cm2 onto Matrigel-coated 6-well plates. From DIV 10 to 20, cultures were supplemented with SHH (200 ng/mL in 0.1% (w/v) BSA in 1X PBS; R&D, Minneapolis, MN, USA) and Purmorphamine (1 µM in DMSO; Sigma, St. Louis, MO, USA). Half-media was changed every other day afterward. On DIV 20, cultures were passaged using 0.5 mM EDTA for 2 min at 37 °C, as we no longer observed issues with survivability with EDTA treatment, and pipetted up and down 10–15 times using P1000 mechanical pipette then replated onto Poly-L-Ornithine (15 µg/mL in water; Sigma, St. Louis, MO, USA) and Laminin (5 µg/mL in PBS; Corning) coated 24-well plates (Greiner Bio-One) at 2.11E5 cells/cm2. Half-media was changed the next day and then every other day afterward. From DIV 20 to 45, media was supplemented with only BDNF (10 ng/mL in 0.1% BSA (w/v); Peprotech) to aid neuronal maturation and survival. Cells were maintained in a humid incubator at 37 °C with 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA extraction, total RNA was extracted as previously described [109]. In brief, hMSN-like cultures were washed 1 time in PBS. Total RNA was isolated using Direct-zol RNA MicroPrep Kit (Zymo Research) according to the manufacturer’s protocol. RNA samples were collected in 2 mL RNAse-free tubes and chilled on ice throughout the procedure. Total RNA was then used for RNA-seq.
RNA libraries for RNA-seq were prepared by Genewiz/Azenta.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
CLC Workbench 23.0.2
Sequence reads were trimmed for adaptor sequence/low-quality sequence using CLC genomic benchwork (parameter - Quality limit: 0.05)
Trimmed sequence reads were mapped to ensembl v97 hg38 using CLC genomic benchwork (default settings)
Read count extraction and normalization were performed using CLC genomic benchwork.
Differential expression was performed using CLC genomic benchwork using default settings.
Assembly: hg38
Supplementary files format and content: xlsx files include TPM and RPKM normalized values and raw counts for each sample.
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| Submission date |
Jul 06, 2023 |
| Last update date |
Jul 09, 2023 |
| Contact name |
Ryan W Tam |
| Phone |
919-515-3352
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| Organization name |
North Carolina State University
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| Department |
Chemical and Biomolecular Engineering
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| Lab |
Keung Lab
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| Street address |
840 Main Campus Drive, Building Partners II, Room 3700
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| City |
Raleigh |
| State/province |
North Carolina |
| ZIP/Postal code |
27603 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE236684 |
Profiling transcriptomic responses of human stem cell-derived medium spiny neuron-like cells to exogenous phasic and tonic neurotransmitters |
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| Relations |
| BioSample |
SAMN36344961 |
| SRA |
SRX20928721 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7567896_AA-pre-AA-dose-5-18-H1-MSN-1-mM-R1_GE_.xlsx |
8.0 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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