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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 10, 2024 |
| Title |
B9_CD05_T/T_Clone2 |
| Sample type |
SRA |
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| Source name |
iPS-derived glutamatergic neuron and primary mouse astrocyte
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| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
tissue: iPS-derived glutamatergic neuron and primary mouse astrocyte
cell line: CD05
cell type: human neuron/mouse astrocyte
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| Treatment protocol |
Reverse transfection was used for SNP editing. gRNAs were cloned to CROPseq-Guide-Puro vector (Addgene #86708) [Datlinger P, nature method, 2017]. Vector pSpCas9(BB)-2A-Puro (Addgene #62988) was used to express Cas9 [Ran FA, nature protocol, 2013]. Vector pSUPERIOR.puro-shp53 (Addgene #38035) was used during the transfection to transiently inhibit p53 and increase editing efficiency [Ihry RJ, nature method, 2018]. In day 1, iPSC was accutased (07920, StemCell) into single cell, then centrifuged, and placed in a 15ml centrifuge tube at 4-6 × 105/1.8ml with 10 μM ROCK inhibitor (1254/1, R&D Systems). Lipofectamine stem reagent (STEM00001, Thermofisher) was used for transfections. In preparation stage, DNA including pSpCas9(BB)-2A-Puro (0.5 μl from 1 μl/ug stock), CROPseq-Guide-Puro vector (1 μl from 1μl/ug stock) pSUPERIOR.puro-shp53 vector (1 μl from 1μl/ug stock) and ssODNs (6 μl from 100uM stock) was mixed into 100 μl Opti-MEM media (31985062, Thermofisher) in 1.5ml tube; another 1.5 ml tube was filled with 8 μl Lipofectamine stem reagent and 100 μl Opti-MEM media. Two 1.5 ml tubes were combined and incubated for 10 minutes, then added in to iPSC single cell suspension, gently mixed well and plated into one well of 6 well plate. After 16 hours post transfection, all medium was carefully refreshed with 0.25 ug/ml puromycin and 10 μM ROCK inhibitor. After 48 hours post transfection, medium was refreshed with same reagents. After 72 hours post transfection, all puromycin was removed, and the cells were covered new medium with 5 μM ROCK inhibitor. In the next 2-3 day, half medium was refreshed without ROCK inhibitor in order to gradually reduce the ROCK inhibitor in the culture system. Once the cell colonies were formed and looked health, ROCK inhibitor was completely removed by refreshing the entire medium. After around 14 days post-transfection, colonies were individually picked into 96-well plates. DNA from a small fraction of cells in each colony was then extracted (QE09050, Epicentre) and used Sanger sequencing. Subcloning with low density (2-5000 cells/6 cm dish) was carried out if the colony was not pure. Following successful editing, three genomic positions of gRNA off-targets were selected based on their highest rankings and analyzed using Sanger sequencing
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| Growth protocol |
The hiPSCs were maintained in mTeSRPlus (StemCell #100-0276) with primocin (Invitrogen #ant-pm-1) on tissue culture plates coated with matrigel (Fisher Scientific #08-774-552) or geltrex (Fisher Scientific #A1413202) throughout the mutagenesis process. Mouse astrocyte were maitained in DMEM (Thermofisher: 10569010) with FBS (Thermofisher: A5209501) and primocin The Institutional Review Board (IRB) of NorthShore University HealthSystem approved study.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Qiagen RNEasy mini spin columns and the concentrations verified by NanoDrop
Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification.Sequencing was performed on NovaX with 2x150 bp paired read
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
G15
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| Data processing |
Raw fastq files (pair-end) were mapped to concatenated reference from human (GRCh38) and mouse (GRCm39) by Salmon (v0.11.3)
Counts were collected from the output of each Salmon.
Assembly: hunan GRCh38 and mouse GRCm39
Supplementary files format and content: FASTQ
Supplementary files format and content: tab-delimited text included raw counts per sample
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| Submission date |
Jun 05, 2024 |
| Last update date |
Jun 10, 2024 |
| Contact name |
Jubao Duan |
| E-mail(s) |
jduan69@gmail.com
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| Phone |
(224) 364-7564
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| Organization name |
NorthShore University HealthSystem
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| Department |
Center for Psychiatric Genetics
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| Lab |
Unit of Functional Genomics in Psychiatry
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| Street address |
1001 University Place
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| City |
Evanston |
| State/province |
IL |
| ZIP/Postal code |
60201 |
| Country |
USA |
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| Platform ID |
GPL16512 |
| Series (1) |
| GSE269153 |
Alzheimer’s Disease Protective Allele of CLU Promotes Neuron Excitability, Neuron-glia Lipid Transfer and Astrocytic Lipid Droplets via neuronal CLU |
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| Relations |
| BioSample |
SAMN41693255 |
| SRA |
SRX24811836 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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