|
| Status |
Public on Feb 07, 2019 |
| Title |
001428 Env Naive Plasmid DNA Library |
| Sample type |
SRA |
| |
|
| Source name |
Synthetic coding sequences
|
| Organism |
Human immunodeficiency virus 1 |
| Characteristics |
protein: Env (gp160)
sort gate: NA
# collected cells: NA
|
| Treatment protocol |
Env sequence variants were cloned into pCEP4 (Invitrogen) containing a chimeric intron in the 5' UTR. Cells were transfected with the 001428(753)-VC plasmid DNA library under conditions that typically give no more than one sequence variant per cell. 24 hours post-transfection, cells were stained with 2 nM PG16 (secondary: APC-conjugated anti-human IgG Fc) to detect Env expression. On a BD FACS Aria II, single cells were gated by FSC/SSC properties, and propidium iodide-positive dead cells and autofluorescent cells in the Pacific Blue channel were excluded. From the PG16-positive population, the 15 % of cells with highest and lowest BiFC signals were collected.
|
| Growth protocol |
Expi293F cells with the CXCR4 gene knocked out using genome editing, and stably expressing the MA domain of Gag fused to VN. Cells were cultured in Expi293 Expression Medium (Life Technologies).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total RNA was harvested from sorted cells using GeneJet RNA Purification Kit (Thermo Scientific) and cDNA was prepared using Accuscript Hi-Fi (Agilent Genomics) primed with EBV reverse primer (GTGGTTTGTCCAAACTCATC).
In a first round of PCR, the 001428 coding sequence was amplified using oligonucleotides with complementary overhangs for annealing to Illumina sequencing primers.
Primer pair for gene-specific amplification: Illumina_001428VC_1996_for (TCTTTCCCTACACGACGCTCTTCCGATCTTTGGTCATGGTTTGATATCTC) and Illumina_001428VC_2277_rev (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTTCTTCTGCTTGTCTCCAG)
In a second round of PCR, Illumina adaptors and experiment-specific barcodes were added.
Primer pair for adding I5 and I7 Illumina adaptors: MiSeq_Start_Adaptamer (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT) and MiSeq_Index_Adaptamer (CAAGCAGAAGACGGCATACGAGAT-6nt-barcode-GTGACTGGAGTTCAGACGTGTGCTCTTC)
Amplicons were deep sequenced on a MiSeq using a 2x300nt PE v3 paired end protocol.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
plasmid DNA, Naive Env SSM library (strain 001428, a.a. N677-L753 )
|
| Data processing |
Data was analyzed using Enrich: http://depts.washington.edu/sfields/software/enrich/
Fuser script: python Paired_read_fuser.py --path output/ --read1 001428_sample_R1.fastq.bz2 --read2 001428_sample_R2.fastq.bz2 --wtseq AATTGGCTCTGGTATATAAAAATTTTCATAATGATAGTAGGTGGGTTGATTGGTCTTCGCATCATTTTTGCGGTATTGTCTATCGTCAACCGAGTAAGACAGGGGTATTCCCCATTGTCTTTCCAAACATTGACCCCAAACCCGACTGGCCCCGACAGACTCGGGAGAATCGAAGAAGAGGGAGGTGAGCAGGATAGAGATAGGAGCGTGAGGCTTGTGAACGGTTTCCTG --read1_overlap_start 22 --read1_overlap_end 253 --read2_overlap_start 42 --read2_overlap_end 273 --paired_mismatch_threshold 301 --mode B
Aligner script: python Fused_read_aligner.py --path output/ --infile 001428_sample_R1.fast_B_qc1 --referenceDNA AATTGGCTCTGGTATATAAAAATTTTCATAATGATAGTAGGTGGGTTGATTGGTCTTCGCATCATTTTTGCGGTATTGTCTATCGTCAACCGAGTAAGACAGGGGTATTCCCCATTGTCTTTCCAAACATTGACCCCAAACCCGACTGGCCCCGACAGACTCGGGAGAATCGAAGAAGAGGGAGGTGAGCAGGATAGAGATAGGAGCGTGAGGCTTGTGAACGGTTTCCTG --referenceAA NWLWYIKIFIMIVGGLIGLRIIFAVLSIVNRVRQGYSPLSFQTLTPNPTGPDRLGRIEEEGGEQDRDRSVRLVNGFL --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode B
MapCounts script: python mapCounts.py --path output/ --infile 001428_sample_R1.fast_R1_qc1_PRO_qc2
MapRatios script: python mapRatios.py --path ratios_directory/ --templatepath /deepseq_scripts/r_deepseq_scripts/ --infile2 mapcounts_library --infile1 mapcounts_sorted
MapParts script: python mapParts.py --path ratios_directory/ --infile mapratios_sorted_library --mode mutations:1
Unlink script: python mapUnlink.py --path ratios/ --infile mapratios_sorted_library.m1 --type protein --mode ratios --size 77
In Excel, the log(base2) enrichment ratio of the wildtype sequence was subtracted from the log(2) enrichment ratios for all single mutations.
Genome_build: Not applicable
Supplementary_files_format_and_content: Excel spreadsheet of log(base2) enrichment ratios for each single amino acid substitution. Also includes the frequency of each mutation in the naïve plasmid library.
|
| |
|
| Submission date |
Feb 06, 2019 |
| Last update date |
Feb 12, 2019 |
| Contact name |
Erik Procko |
| E-mail(s) |
procko@illinois.edu
|
| Phone |
217-300-1454
|
| Organization name |
University of Illinois
|
| Department |
Biochemistry
|
| Lab |
RAL 318G
|
| Street address |
601 S Goodwin Ave
|
| City |
Urbana |
| State/province |
IL |
| ZIP/Postal code |
61801 |
| Country |
USA |
| |
|
| Platform ID |
GPL23868 |
| Series (1) |
| GSE126136 |
A focused deep mutational scan on the Env transmembrane and proximal cytosolic regions reveals an absence of sequence features for MA association |
|
| Relations |
| BioSample |
SAMN10880112 |
| SRA |
SRX5338806 |
