|
| Status |
Public on Jun 16, 2022 |
| Title |
select_wt_replicate2_tile5 |
| Sample type |
SRA |
| |
|
| Source name |
Yeast cells
|
| Organism |
synthetic construct |
| Characteristics |
host yeast strain: hxk1deltahxk2deltaglk1delta
selection: yes
molecule subtype: plasmid
|
| Growth protocol |
Following transformation, yeast cells were grown in SC-URA galactose until saturation. Cells were either pelleted and stored at -20C before plasmid DNA extraction to serve as the nonselect condition or used for selection. For selection of GCK activity, cells were grown on solid SC-URA 0.2% glucose for three days at 30C. After selection, cells were pelleted and stored at -20C prior to plasmid DNA extraction to serve as the post-selection samples (select).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each condition (select/nonselect, library/wt, replicate1/replicate2), 9 OD units of cells were used for plasmid DNA extraction using the ChargeSwitch Plasmid Yeast Mini kit (Invitrogen) according to the manufacturer's instructions.
The three regional libraries spanning the GCK sequence (aa 2-171, 172-337, 338-466) were constructed by pooled mutagenesis using NNK-degenerate oligos.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
Amino acid positions 138-171
|
| Data processing |
Library strategy: Tile-Seq
Libraries were sequenced on a NextSeq 550 instrument (Illumina) and base called using the instriment's Real Time Analysis software.
Sequencing reads were converted to fastq format and demultiplexed using BaseSpace Sequence Hub.
Reads were alligned and variants called using the TileSeq mutation count pipeline (https://github.com/RyogaLi/tileseq_mutcount) version 0.5.9
Variant calls from the TileSeq mutation count pipeline were then used for calling protein-level amino acid changes and for calculating the enrichment of variants using the TileSeqMave pipeline (https://github.com/jweile/tileseqMave) version 0.6.0.9000
reference sequence: sequence.txt on the series record contains the sequence used for read alignment.
|
| |
|
| Submission date |
Mar 17, 2022 |
| Last update date |
Jun 16, 2022 |
| Contact name |
Sarah Gersing |
| E-mail(s) |
sarah.gersing@bio.ku.dk
|
| Organization name |
University of Copenhagen
|
| Street address |
Ole Maaløes vej 5
|
| City |
Copenhagen N |
| State/province |
- |
| ZIP/Postal code |
2200 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL27609 |
| Series (1) |
| GSE198878 |
Multiplexed assessment of human glucokinase variant activity |
|
| Relations |
| BioSample |
SAMN26750663 |
| SRA |
SRX14486758 |
