|
| Status |
Public on Oct 30, 2023 |
| Title |
M4 + L1/2_biol, rep2 |
| Sample type |
SRA |
| |
|
| Source name |
HEK293T-GFP
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T-GFP
cell type: Human Embyonic Kidney Cells with GFP
treatment: Lipid-based transfection of plasmids encoditing base editor M4, GFP-spcific gRNA L1 and GFP-specific gRNA L2 , respectively.
time: Cells harvest 72 h post transfection
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were harvested and genomic DNA was extracted using GeneJet Genomic DNA Purification Kit (Thermo Fisher) following manufacturers protocol. Concentration of isolated gDNA was measured with Nanodrop2000 and stored until further use at -80 °C
Targeted amplicon deep sequencing was performed. Locus of interest was amplified through PCR with barcoded Primers for multiplexing. In total 100 ng gDNA were used together with 0.5 µM each forward and reverse primer and 0.2 µM dNTPs and 0.2 µl phusion polymerase (Thermo Fisher). In total three independent PCR reactions were merged. All barcoded amplicons were pooled in an equal molar ratio.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Sequel II |
| |
|
| Data processing |
To analyse base editing outcome the webtool CRISPResso2 (https://crispresso.pinellolab.partners.org) was used. Fastq.gz files were uploaded together with the reference sequenc and the gRNA sequences. Option Base Editing was choosen and default settings were used.
Supplementary files format and content: Selected_nucleotide_percentage_table_around_sgRNA_XYZ.txt (Txt file given by CRISPResso2; shows all possible nucleotide changes in percentage within the protospacer sequence as well as 10 nts +/- of protospacer.)
Supplementary files format and content: Modification_count_vectors.txt (Txt file given by CRISPResso2; shows percentage of deletions, insertions and substitions for each position of the analyzed sequenced)
Supplementary files format and content: CRISPResso_quantification_of_editing_frequency.txt (Txt file given by CRISPResso2; overall read number per position and the overall substitution and deletion frequency )
Supplementary files format and content: Nucleotide_percentage_table.txt (Txt file given by CRISPResso2; shows all possible nucleotide changes in percentage of the whole input sequence)
|
| |
|
| Submission date |
Jun 20, 2022 |
| Last update date |
Oct 30, 2023 |
| Contact name |
Julian Dirk Alexander Weischedel |
| E-mail(s) |
weischedel@immunology.uzh.ch
|
| Organization name |
University of Zurich
|
| Street address |
Winterthurerstrasse 190
|
| City |
Zurich |
| ZIP/Postal code |
8057 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL28352 |
| Series (1) |
| GSE206445 |
Base editor induced mutation spectrum in GFP, human ATP1a1, human TP53BP1 and murine MyoD |
|
| Relations |
| BioSample |
SAMN29207062 |
| SRA |
SRX15797361 |
