|
| Status |
Public on Jan 11, 2023 |
| Title |
pOD49_PAM_lib_deplete_3 |
| Sample type |
SRA |
| |
|
| Source name |
Plasmid DNA from Liquid culture
|
| Organism |
Escherichia coli |
| Characteristics |
treatment: Control 2
|
| Treatment protocol |
Prior to extraction the cells were harvested by centrifugation at 4,000 g for 15 min.
|
| Growth protocol |
The cultures containing the plasmid library and either the nuclease-expressing or the control plasmid were grown in LB with antibiotic selection and IPTG as well as L-arabinose to induce nuclease and crRNA expression. The cultures were grown for 13 hours at 37C with constant shaking.
|
| Extracted molecule |
other |
| Extraction protocol |
Plasmids were isolated using ZymoPURE II Plasmid Midiprep Kit.
Libraries were constructed by amplifying plasmid DNA region with primers containing Illumina adapters for 25 cycle using Q5 HS High-Fidelity 2X Master Mix. Following amplification, samples were cleaned up with AMPure XP beads. These dsDNAs were then indexed using Nextera barcodes 501, 502, and 707, 708.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
Control plasmid library without SuCasΩ replicate 3
|
| Data processing |
Library strategy: PAM-assay
Illumina reads were quality checked and trimmed using BBDuk.
Forward sequnces in SuCasΩ depleted library sample 1 were extracted as follows: grep 'TCACC[TCAG][TCAG][TCAG][TCAG][TCAG]' pOD4_PAM_lib_deplete_2_S1_R2_001_trimmed.fasta | cut -c 60-64 | sort | uniq -c | sort -nr | less > pOD4_PAM_lib_deplete_2_S1_R2_001_trimmed_count_5prime.txt
Forward sequnces in SuCasΩ depleted library sample 2 were extracted as follows: grep 'TCACC[TCAG][TCAG][TCAG][TCAG][TCAG]' pOD4_PAM_lib_deplete_3_S2_R2_001_trimmed.fasta | cut -c 60-64 | sort | uniq -c | sort -nr | less > pOD4_PAM_lib_deplete_3_S2_R2_001_trimmed_count_5prime.txt
Forward sequnces in SuCasΩ control library sample 1 were extracted as follows: grep 'TCACC[TCAG][TCAG][TCAG][TCAG][TCAG]' pOD49_PAM_lib_deplete_2_S3_R2_001_trimmed.fasta | cut -c 60-64 | sort | uniq -c | sort -nr | less > pOD49_PAM_lib_deplete_2_S3_R2_001_trimmed_count_5prime.txt
Forward sequnces in SuCasΩ control library sample 2 were extracted as follows: grep 'TCACC[TCAG][TCAG][TCAG][TCAG][TCAG]' pOD4_PAM_lib_deplete_3_S2_R2_001_trimmed.fasta | cut -c 60-64 | sort | uniq -c | sort -nr | less > pOD4_PAM_lib_deplete_3_S2_R2_001_trimmed_count_5prime.txt
Reverse sequnces in SuCasΩ depleted library sample 1 were extracted as follows: grep 'CTCCA[TCAG][TCAG][TCAG][TCAG][TCAG]' pOD4_PAM_lib_deplete_2_S1_R1_001_trimmed.fasta | cut -c 71-75 | sort | uniq -c | sort -nr | less > pOD4_PAM_lib_deplete_2_S1_R1_001_trimmed_count_3prime.txt
Reverse sequnces in SuCasΩ depleted library sample 2 were extracted as follows: grep 'CTCCA[TCAG][TCAG][TCAG][TCAG][TCAG]' pOD4_PAM_lib_deplete_3_S2_R1_001_trimmed.fasta | cut -c 71-75 | sort | uniq -c | sort -nr | less > pOD4_PAM_lib_deplete_3_S2_R1_001_trimmed_count_3prime.txt
Reverse sequnces in SuCasΩ control library sample 1 were extracted as follows: grep 'CTCCA[TCAG][TCAG][TCAG][TCAG][TCAG]' pOD49_PAM_lib_deplete_2_S3_R1_001_trimmed.fasta | cut -c 71-75 | sort | uniq -c | sort -nr | less > pOD49_PAM_lib_deplete_2_S3_R1_001_trimmed_count_3prime.txt
Reverse sequnces in SuCasΩ control library sample 2 were extracted as follows: grep 'CTCCA[TCAG][TCAG][TCAG][TCAG][TCAG]' pOD49_PAM_lib_deplete_3_S4_R1_001_trimmed.fasta | cut -c 71-75 | sort | uniq -c | sort -nr | less > pOD49_PAM_lib_deplete_3_S4_R1_001_trimmed_count_3prime.txt
The depleted sequenes were determined using the following formula: (Total number of control reads/Total number of depleted reads)×(Number depleted reads per sequence/number control reads per cequence)
Supplementary_files_format_and_content: fasta: file containing quality-checked and trimmed sequences
Supplementary_files_format_and_content: txt: file containing sequence counts
|
| |
|
| Submission date |
Jun 21, 2021 |
| Last update date |
Jan 11, 2023 |
| Contact name |
Chase L Beisel |
| Organization name |
Helmholtz Institute for RNA-Based Infection Research
|
| Lab |
RSYN
|
| Street address |
Josef-Schneider-Str. 2
|
| City |
Wurzburg |
| ZIP/Postal code |
97080 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16085 |
| Series (2) |
| GSE178530 |
Deep sequencing data from PFS screen of SuCas12a2 CRISPR-Cas nuclease |
| GSE178536 |
Cas12a2 elicits abortive infection through RNA-triggered destruction of dsDNA |
|
| Relations |
| BioSample |
SAMN19795483 |
| SRA |
SRX11187501 |
