|
| Status |
Public on Sep 17, 2024 |
| Title |
V4617 |
| Sample type |
SRA |
| |
|
| Source name |
iPS Cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line_barcode: V000874617
tissue: iPS Cells
cell line: CD14
cell type: iPSC
age: 33
Sex: Female
ancestry: EA
lof target_gene: Negative Control
chromosome of_gene: N/A
lof variant_coordinate_(hg38_bp_position): N/A
variant type: N/A
crispr type: CBE4max
passage #: 21_4
|
| Treatment protocol |
The cell culture, DNA base editing/sgRNA transfection, and cell sorting for LoF mutagenesis were performed in batches, each containing 23 genes and a non-transfected control (NTC) on a 24-well plate format. The base editor system (pEF-AncBE4max, pEF-BFP, and pDT-sgRNA) contains a reporter gene that makes cells that have undergone C to T editing turn from blue to green . LipofectamineSTEM was used for cell transfection. After cell transfection for 72 hrs, single hiPSC from the post-transfection culture were sorted into 96-well plates with one cell per well using a BD FACSAria Fusion Flow Cytometer in the presence of CEPT cocktail (1:10,000 chroman 1, emricasan, and transISRIB; 1:1,000 polyamine supplement) (Tristan et al., 2023). The sorted single cells were cultured on a 96-well plate with media changes every other day for 10-14 days until colonies appeared with an appropriate size to pick for Sanger sequencing genotyping.
|
| 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. The Institutional Review Board (IRB) of NorthShore University HealthSystem approved study.
|
| 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
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq X |
| |
|
| Description |
sample_id in count_matrix: V4617
|
| Data processing |
Raw fastq files (pair-end) were aligned to human GRCh38.p13 genome using STAR 2.7.11 with GENCODE 35 annotations.
Counts were collected from the output of each STAR run and merged into one count matrix
Assembly: hunan GRCh38.p14 (hg38)
Supplementary files format and content: FASTQ
Supplementary files format and content: tab-delimited text included raw counts per sample
|
| |
|
| Submission date |
Mar 26, 2024 |
| Last update date |
Sep 17, 2024 |
| Contact name |
Jubao Duan |
| E-mail(s) |
jduan69@gmail.com
|
| Phone |
(224) 364-7564
|
| Organization name |
NorthShore University HealthSystem
|
| Department |
Center for Psychiatric Genetics
|
| Lab |
Unit of Functional Genomics in Psychiatry
|
| Street address |
1001 University Place
|
| City |
Evanston |
| State/province |
IL |
| ZIP/Postal code |
60201 |
| Country |
USA |
| |
|
| Platform ID |
GPL34281 |
| Series (1) |
| GSE262442 |
Scaled and efficient derivation of loss-of-function alleles in risk genes for neurodevelopmental and psychiatric disorders in human iPSCs |
|
| Relations |
| BioSample |
SAMN40615126 |
| SRA |
SRX24059825 |
