|
| Status |
Public on Jul 31, 2019 |
| Title |
Output 1 High expression Rep3 |
| Sample type |
SRA |
| |
|
| Source name |
Deep mutational scanning
|
| Organism |
synthetic construct |
| Characteristics |
mutant protein expression level: high
|
| Growth protocol |
E.coli BW27783 MK01 strain (Kogenaru and Tans, BMC 2014) bacteria were grown in LB media (containing ampicilin) with arabinose and glucose to induce protein expression at 37ºC for 4 hours in total. Cells before sorting and after sorting were centrifuged and the pellets were stored at -20ºC until DNA extraction
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
All the cell pellets were first thawed on ice, and plasmid preparation was performed using the QIAGEN miniprep kit on column.
The mutagenized region was amplified using barcoded PCR primers (in order to multiplex, PCR primers carried 8nt barcodes at the 5 prime to identify each sample) for 25 cycles using hot start Phusion polymerase in 50μl reactions, following the manufacturers’ instruction. PCR products were purified using the E-gel 2% size-select system. (Invitrogen) to remove smaller fragments. Samples were mixed equimolarly and and stored at -20 degrees, and sent to EMBL Genecore on dry ice, where PCR-free sequencing library was prepared and sequenced.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
PCR amplicon
from plasmid library
|
| Data processing |
Library strategy: Deep mutational scanning
1. Pair end reads were demultiplexed with Sabre (v 1.000, with parameter -m 2 -pe)
2. Merging reads were performed with PEAR software ( v 0.9.6, with parameters : -m 214 -v 20 -n 214 -j 4 for samples 1-6,10-18; for the sample 7-9, with the parameter -m 230 -v 4 -n 230 -j 4).
3. fastx_reverse_complement tool from FASTX-Toolkit (v0.0.13) is used to complement the sequences whenever necessary.
4. The primer sequences were trimmed using the seqtk tool (https://github.com/lh3/seqtk, with parameters -b 19 e-18 for samples 1-6,10-19; and -b 30 e- 30).
5.The number of occurrences of each variant was counted with fastx_collapser from FASTX Toolkit (v 0.0.13) and custom python script.
Supplementary_files_format_and_content: Text file with all merged read count for each variant in each sample
|
| |
|
| Submission date |
Nov 21, 2018 |
| Last update date |
Aug 02, 2019 |
| Contact name |
Xianghua Li |
| E-mail(s) |
xianghua.li@crg.eu
|
| Organization name |
Centre for Genomic Regulation
|
| Street address |
carrer Dr. Aiguader 88, PRBB building
|
| City |
Barcelona |
| State/province |
Barcelona |
| ZIP/Postal code |
08003 |
| Country |
Spain |
| |
|
| Platform ID |
GPL19604 |
| Series (1) |
| GSE122806 |
Changes in gene expression predictably shift and switch genetic interactions |
|
| Relations |
| BioSample |
SAMN10459563 |
| SRA |
SRX5052508 |
