|
| Status |
Public on Apr 26, 2021 |
| Title |
Pulldown Med15 LibAB replicate3 |
| Sample type |
SRA |
| |
|
| Source name |
Med15 LibAB replicate3
|
| Organism |
synthetic construct |
| Characteristics |
pulldown: Med15
|
| Treatment protocol |
In vitro expressed protein library input was incubated with the bait-coated beads in binding buffer with salmon sperm DNA and BSA at 100 µL total volume for 8 min with mixing. Afterward, the beads were separated on a magnet, briefly washed once with cold binding buffer, and then immediately separated on a magnet again. cDNA from bound fragments was eluted by suspending the beads in 10 mM Tris pH 8.0 with proteinase K (NEB) and incubated for 30 min at room temperature. Proteinase K was heat inactivated at 95C for 10 min, beads were separated on a magnet, and the eluate was collected.
|
| Growth protocol |
N/A
|
| Extracted molecule |
total RNA |
| Extraction protocol |
N/A
The cDNA region encoding the variable protein fragment was PCR-amplified for 11-20 cycles using Phusion polymerase with PCR primers that added flanking sequences corresponding to the standard Illumina sequencing primers. After a column cleanup, a second PCR was performed with primers that added sample-specific barcodes and the P5 and P7 sequences for Illumina sequencing.
OTHER:sequncing of designed oligo library
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
cDNA of mRNA-tagged protein
|
| Data processing |
UMI deduplication using umi_tools 1.0.0
cutadapt 1.18 was used to discard reads without matching paired-end sublibrary sequencing primers and trim the primers in reads with matching primers
bwa-mem 0.7.17-r1188 was used to perform a first-pass alignment of reads to the DNA fragment library
Imperfectly mapped read pairs (i.e., those without paired read SAM flags of 99 and 147) were re-mapped to the library sequence with minimal edit distance
Supplementary_files_format_and_content: Excel file containing counts of reads aligned to each protein fragment
|
| |
|
| Submission date |
Apr 22, 2021 |
| Last update date |
Apr 26, 2021 |
| Contact name |
Adrian L Sanborn |
| E-mail(s) |
a@adriansanborn.com
|
| Organization name |
Stanford University
|
| Street address |
299 Campus Drive West
|
| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
|
| Platform ID |
GPL27609 |
| Series (2) |
| GSE173152 |
Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator (Pulldown) |
| GSE173156 |
Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator |
|
| Relations |
| BioSample |
SAMN18836528 |
| SRA |
SRX10660622 |
