|
| Status |
Public on May 31, 2024 |
| Title |
HDR template library pDNA Tn5 |
| Sample type |
SRA |
| |
|
| Source name |
HAP1
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HAP1
cell type: near-haploid cells derived from KBM-7
treatment: NA
|
| Treatment protocol |
Cells were transfected via Lipofection with all-in-one pSpCas9+sgRNA+puro constructs with or without KARS1 Exon 2 variant HDR template library containing all possible variants at in-frame codons. Transfected cells were selected with 2.5 ug/mL puromycin for 48 hrs and allowed to recover to 70% confluency. Cells were passaged every 2-3 days for 2 additional passages (p1 and p2).
|
| Growth protocol |
Cells were cultured in IMDM + 10% FBS + 1% pen/strep solution under standard growth conditions in 10 cm cell culture dishes. Cells were passaged by trypsinization every 2-3 days.
|
| Extracted molecule |
other |
| Extraction protocol |
Total genomic DNA was isolated using the Macherey-Nagel NucleoSpin Tissue Mini DNA extraction kit.
Tn5 transposase conjugated with Illumina/Nextera-compatible adapters was used to simultaneously fragment and ligate adapters onto genomic DNA. Resultant fragments were amplified with Illumina i5/i7 universal primers and unique dual index primers in a subsequent barcode PCR reaction.
Tn5 transposase library preparation with Illumina/Nextera-compatible adapter overhangs and dual indices
|
| |
|
| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Tn5 Input HDR template library Rep 1
|
| Data processing |
Paired-end reads were trimmed using Cutadapt 4.1
Trimmed reads were mapped to H. sapiens GRCh38 using Bowtie2 2.2.5
Reads mapping to KARS1 Exon 2 in BAM files were assessed for WT genomic DNA sequence, insertions/deletions, and HDR knock-in using custom Python scripts with Pysam 0.21.0
Called reads were converted to BED file using Bedtools 2.31.0
Assembly: H. sapiens GRCh38 (Bowtie2 GRCh38 no-alt analysis set premade index)
Supplementary files format and content: BAM files containing reads mapped to KARS1 Exon 2 (read category indicated in 'CO' tag)
Supplementary files format and content: .txt files containing counts of reads mapped to KARS Exon 2 by read category
Library strategy: Tn5-Seq
|
| |
|
| Submission date |
May 10, 2023 |
| Last update date |
May 31, 2024 |
| Contact name |
Anthony Antonellis |
| E-mail(s) |
antonell@umich.edu
|
| Organization name |
University of Michigan
|
| Department |
Human Genetics
|
| Lab |
Antonellis
|
| Street address |
1241 Catherine St
|
| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE232151 |
Massively parallel saturation genome editing of an essential mitochondrial targeting sequence (Tn5) |
| GSE232152 |
Massively parallel saturation genome editing of an essential mitochondrial targeting sequence |
|
| Relations |
| BioSample |
SAMN35025813 |
| SRA |
SRX20277639 |
