|
| Status |
Public on Jan 18, 2023 |
| Title |
U266 FBC selection rep 2 |
| Sample type |
SRA |
| |
|
| Source name |
In vitro IgG repertoire
|
| Organism |
Oryctolagus cuniculus |
| Characteristics |
tissue: In vitro IgG repertoire
treatment: Selected against U266 cell line with FBC method
|
| Treatment protocol |
Phage libraries and human cells were incubated in 3% (w/v) Bovine Serum Albumin (BSA), 0.025% (w/v) NaN3, and 1 mM EDTA in PBS. After incubation and 3x PBS washing, phage were eluted using low pH. Output phage were processed with or without the FBC method,, and were then rescued and reamplified in ER2738 bacterial cells, followed by glycerol stock preparation.
|
| Growth protocol |
Phage Fab-antibody repertoires were propagated in ER2738 bacterial cells using VCSM13 helper phage. The following cell lines were all cultured at 37ºC in 5% CO2 in complete medium consisting of RPMI-1640 medium containing 10% (v/v) FBS (BioFluid) and 1x PS: NCI-H929 (H929), U266. HEK293-ROR1-TetOn cells cells were cultured identically, but with DMEM as the base medium, and with or without 100 ng/mL doxycycline to induce ROR1 cell surface expression. Fresh healthy donor PBMCs were bought from AllCells and cryopreserved prior to use with 50% FBS, 10% DMSO in RPMI-1640 medium.
|
| Extracted molecule |
other |
| Extraction protocol |
0.5 mL of the bacterial panning output glycerol stocks was subjected to purified phagemid preparation using the QIAprep Spin Miniprep Kit (Qiagen).
Rabbit VH domains were amplified via a two-step PCR procedure in which Illumina P5 and P7 adapters were added.
Libraries were sequenced on a MiSeq sequencer (Illumina) using MiSeq Reagent Kit v3 (150-cycle) in a single end configuration with reads originating from the P5 primer binding site.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
other |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
plasmid DNA
|
| Data processing |
HCDR3 DNA reads without proper adapters were removed using R.
DNA sequences were aligned to correct antibody reading frame and translated.
Rabbit HCDR3 sequences were extracted using a REGEX recognizing rabbit HCDR3 junctional sequences.
Read counts were compiled and count matrices generated across multiple sequenced libraries.
Differential expression analysis was carried out using DESeq2.
Supplementary files format and content: HCDR3 amino acid counts per sample
Supplementary files format and content: Processed output for DE comparisons after DESeq2 analysis
|
| |
|
| Submission date |
Jan 14, 2023 |
| Last update date |
Jan 19, 2023 |
| Contact name |
Henry Wilson |
| E-mail(s) |
hedawils@gmail.com
|
| Organization name |
The Scripps Research Institute
|
| Lab |
Rader
|
| Street address |
130 Scripps Way
|
| City |
Jupiter |
| State/province |
FL |
| ZIP/Postal code |
33458 |
| Country |
USA |
| |
|
| Platform ID |
GPL28443 |
| Series (1) |
| GSE222897 |
NGS of phage antibody outputs from whole-cell panning |
|
| Relations |
| BioSample |
SAMN32738792 |
| SRA |
SRX19033767 |
