|
| Status |
Public on Jul 31, 2019 |
| Title |
Transport Sorted CT Library; Replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
Synthetic coding sequences
|
| Organism |
Homo sapiens |
| Characteristics |
protein: SERT
sort gate: Top 50% of population for expression (1/200anti-myc) and top 15% of Transporters
# collected cells: 132,000
|
| Treatment protocol |
Cells expressing DNA libraries of SERT mutants (cloned into a modified version of pCEP4, Invitrogen, with an upstream chimeric intron were FACS sorted for expression (the ability of an antibody to bind to an epitope tag in SERT) or transport a flourescent ligand (APP+). Two single site-saturation mutagenesis libraries were created, spanning residues 2-349(NT Library), 350-630 (CT Library).
|
| Growth protocol |
Expi293F cells cultured in Expi293 Expression Medium (Life Technologies)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was harvested from sorted cells using GeneJet RNA Purification Kit (Thermo Scientific) and cDNA prepared using Accuscript Hi-Fi (Agilent Genomics) primed with a gene-specific primer: SERT_NT__rev (__________, NT Library) and EBV_reverse (GTGGTTTGTCCAAACTCATC, CT Library).
In the first round of PCR (20 cycles), the SERT coding sequences were amplified as overlapping fragments of ~700 bp each in the case of the NT Library or ~600bp for the CT library.
Primer pair (NT Library Fragment 1): MiSeq_pCEP4intron_F2 (5`TCTTTCCCTACACGACGCTCTTCCGATCTACGACTCACTATAGGCTAGC3`) and MiSeq_SERT_1080_R (5`GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGAAAAGAAGATTTGCGCGG3`).
Primer pair (NT Library Fragment 2): MiSeq_SERT_191_F (5`TCTTTCCCTACACGACGCTCTTCCGATCTGATACACGGCACAGCATTC3`) and MiSeq_SERT_902_R (5`GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGATGTCTTTACTCCCTTCCAG3`).
Primer pair (NT Library Fragment 3): MiSeq_SERT_426_F (5`TCTTTCCCTACACGACGCTCTTCCGATCTGTATCCCGCTCTTCTACATG3`) and MiSeq_SERT_698_R (5`GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAGTCCTCCTCGCTAATCAG3`).
Primer pair (CTLibrary Fragment 1): MiSeq_SERT_1041_F (5`TCTTTCCCTACACGACGCTCTTCCGATCTGGCAGAAGCTTCTTGAAACC3`) and MiSeq_SERT_1592_R (5`GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATCAATGCCACGGTCAGAAC3`).
Primer pair (CT Library Fragment 2): MiSeq_SERT_1250_F (5`TCTTTCCCTACACGACGCTCTTCCGATCTGCTGAGATGAGGAACGAAGA3`) and MiSeq_SERT_1808_R (5`GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGCCCAGGATAATTGACCAG3`).
Primer pair (CT Library Fragment 3): MiSeq_SERT_1376_F (5`TCTTTCCCTACACGACGCTCTTCCGATCTGATGCTGATAACCCTTGG3`) and MiSeq_pCEP4_R (5`GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTATCATGTCTGGATCCGG3`).
In a second round of PCR (15 cycles), 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 with MiSeq v3 and HiSeq 2500 instruments using a 2x250 nt paired end protocol, though data is analyzed as single reads.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
Deep mutational scan of SERT for transport
|
| Data processing |
Data was analyzed using Enrich: http://depts.washington.edu/sfields/software/enrich/
Fuser Script: Fragmnt NT A read1: python2.7 Paired_read_fuser.py --path NT_fragA_R1/ --read1 SERT_SCIT_R1.fastq.bz2 --read2 SERT_SCIT_R1.fastq.bz2 --wtseq GAGACCACCCCCCTGAACAGTCAAAAGCAACTCTCTGCCTGTGAGGATGGAGAGGACTGTCAGGAGAATGGCGTTCTTCAAAAGGTTGTACCCACCCCTGGAGATAAGGTGGAAAGCGGCCAGATTTCCAACGGCTACAGCGCCGTGCCATCACCTGGAGCCGGGGATGATACACGGCACAGCATTCCCGCAACCACCACGACACTG --read1_overlap_start 29 --read1_overlap_end 236 --mode R1
Fuser_library_NT_fragA_R2.sh: python2.7 Paired_read_fuser.py --path NT_fragA_R2/ --read1 SERT_SCIT_R2.fastq.bz2 --read2 SERT_SCIT_R2.fastq.bz2 --wtseq TTCAAGGGAATCGGATACGCAATCTGTATAATCGCGTTCTATATTGCATCATATTACAACACCATCATGGCCTGGGCCCTTTATTACTTGATCAGCAGTTTCACCGACCAATTGCCGTGGACGAGCTGTAAGAACTCATGGAATACCGGGAACTGTACCAACTACTTCTCTGAGGACAGCTCAGGAGGTAGCGGGGGTGAACAG --read2_overlap_start 23 --read2_overlap_end 227 --mode R2
Fuser_library_NT_fragB_R1.sh: python2.7 Paired_read_fuser.py --path NT_fragB_R1/ --read1 SERT_SCIT_R1.fastq.bz2 --read2 SERT_SCIT_R1.fastq.bz2 --wtseq GCAACCACCACGACACTGGTTGCAGAGCTTCACCAGGGCGAGAGGGAGACGTGGGGCAAAAAGGTAGATTTTCTGCTTTCCGTGATCGGTTACGCGGTCGATCTGGGGAACGTGTGGAGGTTCCCTTACATTTGTTACCAAAACGGAGGGGGTGCCTTCTTGCTGCCCTACACAATTATGGCGATTTTTGGGGGTATCCCGCTCTTCTACATGG --read1_overlap_start 21 --read1_overlap_end 235 --mode R1
Fuser_library_NT_fragB_R2.sh: python2.7 Paired_read_fuser.py --path NT_fragB_R2/ --read1 SERT_SCIT_R2.fastq.bz2 --read2 SERT_SCIT_R2.fastq.bz2 --wtseq CAGAAGCTGATTAGCGAGGAGGACTTGGGCAGTCCAGGCGGAAGCGGCAACATCACTTGGACACTCCATAGTACGAGCCCCGCAGAGGAGTTTTACACCAGGCACGTACTGCAGATTCATAGGAGCAAGGGTCTGCAAGACCTGGGCGGTATTAGCTGGCAGCTCGCATTGTGCATAATGTTGATCTTTACGGTGATCTATTTCTCT --read2_overlap_start 20 --read2_overlap_end 227 --mode R2
Fuser_library_NT_fragC_R1.sh: python2.7 Paired_read_fuser.py --path NT_fragC_R1/ --read1 SERT_SCIT_R1.fastq.bz2 --read2 SERT_SCIT_R1.fastq.bz2 --wtseq GAACTGGCCTTGGGTCAGTACCACCGGAACGGCTGCATTAGCATTTGGCGCAAGATATGCCCCATTTTCAAGGGAATCGGATACGCAATCTGTATAATCGCGTTCTATATTGCATCATATTACAACACCATCATGGCCTGGGCCCTTTATTACTTGATCAGCAGTTTCACCGACCAATTGCCGTGGACGAGCTGTAAG --read1_overlap_start 20 --read1_overlap_end 218 --mode R1
Fuser_library_NT_fragC_R2.sh: python2.7 Paired_read_fuser.py --path NT_fragC_R2/ --read1 SERT_SCIT_R2.fastq.bz2 --read2 SERT_SCIT_R2.fastq.bz2 --wtseq TTTACGGTGATCTATTTCTCTATCTGGAAGGGAGTAAAGACATCCGGCAAGGTGGTGTGGGTGACGGCGACCTTCCCCTACATTATACTCAGCGTGTTGTTGGTGAGGGGGGCAACCCTGCCTGGGGCCTGGCGAGGCGTGCTGTTCTATCTCAAGCCAAACTGGCAGAAGCTTCTTGAAACCGGAGTGTGGATCGACGCG --read2_overlap_start 28 --read2_overlap_end 229 --mode R2
Fuser_library_CT_fragA_R1.sh: python2.7 Paired_read_fuser.py --path CT_fragA_R1/ --read1 SERT_CT_DNA1_R1.fastq.bz2 --read2 SERT_CT_DNA1_R1.fastq.bz2 --wtseq TGGATCGACGCGGCCGCGCAAATCTTCTTTTCCCTCGGTCCCGGCTTCGGGGTCCTTCTGGCATTCGCAAGCTACAACAAGTTCAACAATAACTGCTACCAGGACGCCCTCGTGACAAGTGTAGTGAACTGCATGACCTCTTTCGTGAGCGGGTTTGTGATCTTCACAGTACTCGGATATATGGCTGAGATGAGGAACGAAGAT --read1_overlap_start 26 --read1_overlap_end 230 --mode R1
Fuser_library_CT_fragA_R2.sh: python2.7 Paired_read_fuser.py --path CT_fragA_R2/ --read1 SERT_CT_DNA1_R2.fastq.bz2 --read2 SERT_CT_DNA1_R2.fastq.bz2 --wtseq CTGGATAGCACCTTCGCCGGACTGGAGGGTGTCATCACTGCCGTTCTCGACGAGTTCCCCCATGTGTGGGCCAAGCGACGGGAAAGGTTCGTGCTGGCCGTCGTAATCACATGCTTCTTCGGCTCACTGGTGACCCTTACCTTTGGCGGTGCGTACGTGGTCAAGCTGCTCGAAGAGTACGCCACTGGACCGGCG --read2_overlap_start 35 --read2_overlap_end 230 --mode R2
Fuser_library_CT_fragB_R1.sh: python2.7 Paired_read_fuser.py --path CT_fragB_R1/ --read1 SERT_CT_DNA1_R1.fastq.bz2 --read2 SERT_CT_DNA1_R1.fastq.bz2 --wtseq GTTAGTGAGGTTGCCAAAGACGCTGGCCCGAGCCTGTTGTTCATCACATACGCCGAGGCTATCGCGAACATGCCCGCATCTACTTTCTTCGCCATCATCTTTTTCCTGATGCTGATAACCCTTGGGCTGGATAGCACCTTCGCCGGACTGGAGGGTGTCATCACTGCCGTTCTCGACGAGTTCCCCCATGTGTGGGCCAAG --read1_overlap_start 21 --read1_overlap_end 222 --mode R1
Fuser_library_CT_fragB_R2.sh: python2.7 Paired_read_fuser.py --path CT_fragB_R2/ --read1 SERT_CT_DNA1_R2.fastq.bz2 --read2 SERT_CT_DNA1_R2.fastq.bz2 --wtseq GTTCTGACCGTGGCATTGATCGAGGCCGTGGCCGTAAGCTGGTTTTACGGCATCACGCAGTTCTGTAGGGACGTGAAAGAGATGCTGGGCTTTTCTCCCGGTTGGTTTTGGAGGATTTGTTGGGTAGCAATTAGCCCCCTGTTTCTCCTTTTCATTATTTGCTCTTTTCTGATGTCTCCACCGCAGCTCCGACTGTTTCAGTACAACTACCCC --read2_overlap_start 15 --read2_overlap_end 228 --mode R2
Fuser_library_CT_fragC_R1.sh: python2.7 Paired_read_fuser.py --path CT_fragC_R1/ --read1 SERT_CT_DNA1_R1.fastq.bz2 --read2 SERT_CT_DNA1_R1.fastq.bz2 --wtseq CTGGATAGCACCTTCGCCGGACTGGAGGGTGTCATCACTGCCGTTCTCGACGAGTTCCCCCATGTGTGGGCCAAGCGACGGGAAAGGTTCGTGCTGGCCGTCGTAATCACATGCTTCTTCGGCTCACTGGTGACCCTTACCTTTGGCGGTGCGTACGTGGTCAAGCTGCTCGAAGAGTACGCCACTGGACCGGCG --read1_overlap_start 19 --read1_overlap_end 214 --mode R1
Fuser_library_CT_fragC_R2.sh: python2.7 Paired_read_fuser.py --path CT_fragC_R2/ --read1 SERT_CT_DNA1_R2.fastq.bz2 --read2 SERT_CT_DNA1_R2.fastq.bz2 --wtseq CCCTACTGGTCAATTATCCTGGGCTATTGTATCGGCACCTCAAGTTTCATCTGCATCCCCACATACATCGCATACAGACTGATAATTACGCCCGGTACTTTTAAGGAGCGAATCATTAAAAGCATAACCCCCGAAACCCCCACCGAGATCCCTTGTGGCGATATAAGACTCAATGCGGTC --read2_overlap_start 30 --read2_overlap_end 210 --mode R2
Aligner Script: Fragment NT_A Read 1: python2.7 Fused_read_aligner.py --path NT_fragA_R1/ --infile SERT_library_NT_R1.fast_R1_qc1 --referenceDNA GAGACCACCCCCCTGAACAGTCAAAAGCAACTCTCTGCCTGTGAGGATGGAGAGGACTGTCAGGAGAATGGCGTTCTTCAAAAGGTTGTACCCACCCCTGGAGATAAGGTGGAAAGCGGCCAGATTTCCAACGGCTACAGCGCCGTGCCATCACCTGGAGCCGGGGATGATACACGGCACAGCATTCCCGCA --referenceAA ETTPLNSQKQLSACEDGEDCQENGVLQKVVPTPGDKVESGQISNGYSAVPSPGAGDDTRHSIPA --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner Script: Fragment NT_A Read 2: python2.7 Fused_read_aligner.py --path NT_fragA_R2/ --infile SERT_library_NT_R2.fast_R2_qc1 --referenceDNA TTCAAGGGAATCGGATACGCAATCTGTATAATCGCGTTCTATATTGCATCATATTACAACACCATCATGGCCTGGGCCCTTTATTACTTGATCAGCAGTTTCACCGACCAATTGCCGTGGACGAGCTGTAAGAACTCATGGAATACCGGGAACTGTACCAACTACTTCTCTGAGGACAGCTCAGGAGGTAGCGGGGGTGAACAG --referenceAA FKGIGYAICIIAFYIASYYNTIMAWALYYLISSFTDQLPWTSCKNSWNTGNCTNYFSEDSSGGSGGEQ --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner Script: Fragment NT_B Read 1: python2.7 Fused_read_aligner.py --path NT_fragB_R1/ --infile SERT_library_NT_R1.fast_R1_qc1 --referenceDNA GCAACCACCACGACACTGGTTGCAGAGCTTCACCAGGGCGAGAGGGAGACGTGGGGCAAAAAGGTAGATTTTCTGCTTTCCGTGATCGGTTACGCGGTCGATCTGGGGAACGTGTGGAGGTTCCCTTACATTTGTTACCAAAACGGAGGGGGTGCCTTCTTGCTGCCCTACACAATTATGGCGATTTTTGGGGGTATCCCGCTCTTCTACATGG --referenceAA ATTTTLVAELHQGERETWGKKVDFLLSVIGYAVDLGNVWRFPYICYQNGGGAFLLPYTIMAIFGGIPLFYM --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner Script: Fragment NT_B Read 2: python2.7 Fused_read_aligner.py --path NT_fragB_R2/ --infile SERT_library_NT_R2.fast_R2_qc1 --referenceDNA CAGAAGCTGATTAGCGAGGAGGACTTGGGCAGTCCAGGCGGAAGCGGCAACATCACTTGGACACTCCATAGTACGAGCCCCGCAGAGGAGTTTTACACCAGGCACGTACTGCAGATTCATAGGAGCAAGGGTCTGCAAGACCTGGGCGGTATTAGCTGGCAGCTCGCATTGTGCATAATGTTGATCTTTACGGTGATCTATTTCTCT --referenceAA QKLISEEDLGSPGGSGNITWTLHSTSPAEEFYTRHVLQIHRSKGLQDLGGISWQLALCIMLIFTVIYFS --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner Script: Fragment NT_C Read 1: python2.7 Fused_read_aligner.py --path NT_fragC_R1/ --infile SERT_library_NT_R1.fast_R1_qc1 --referenceDNA GAACTGGCCTTGGGTCAGTACCACCGGAACGGCTGCATTAGCATTTGGCGCAAGATATGCCCCATTTTCAAGGGAATCGGATACGCAATCTGTATAATCGCGTTCTATATTGCATCATATTACAACACCATCATGGCCTGGGCCCTTTATTACTTGATCAGCAGTTTCACCGACCAATTGCCGTGGACGAGCTGTAAG --referenceAA ELALGQYHRNGCISIWRKICPIFKGIGYAICIIAFYIASYYNTIMAWALYYLISSFTDQLPWTSCK --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner Script: Fragment NT_C Read 2: python2.7 Fused_read_aligner.py --path NT_fragC_R2/ --infile SERT_library_NT_R2.fast_R2_qc1 --referenceDNA TTTACGGTGATCTATTTCTCTATCTGGAAGGGAGTAAAGACATCCGGCAAGGTGGTGTGGGTGACGGCGACCTTCCCCTACATTATACTCAGCGTGTTGTTGGTGAGGGGGGCAACCCTGCCTGGGGCCTGGCGAGGCGTGCTGTTCTATCTCAAGCCAAACTGGCAGAAGCTTCTTGAAACCGGAGTGTGGATCGACGCG --referenceAA FTVIYFSIWKGVKTSGKVVWVTATFPYIILSVLLVRGATLPGAWRGVLFYLKPNWQKLLETGVWIDA --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner Script: Fragment CT_A read 1: python2.7 Fused_read_aligner.py --path CT_fragA_R1/ --infile SERT_CT_DNA1_R1.fast_R1_qc1 --referenceDNA TGGATCGACGCGGCCGCGCAAATCTTCTTTTCCCTCGGTCCCGGCTTCGGGGTCCTTCTGGCATTCGCAAGCTACAACAAGTTCAACAATAACTGCTACCAGGACGCCCTCGTGACAAGTGTAGTGAACTGCATGACCTCTTTCGTGAGCGGGTTTGTGATCTTCACAGTACTCGGATATATGGCTGAGATGAGGAACGAAGAT --referenceAA WIDAAAQIFFSLGPGFGVLLAFASYNKFNNNCYQDALVTSVVNCMTSFVSGFVIFTVLGYMAEMRNED --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner Script: Fragment CT_A read 2: python2.7 Fused_read_aligner.py --path CT_fragA_R2/ --infile SERT_CT_DNA1_R2.fast_R2_qc1 --referenceDNA CTGGATAGCACCTTCGCCGGACTGGAGGGTGTCATCACTGCCGTTCTCGACGAGTTCCCCCATGTGTGGGCCAAGCGACGGGAAAGGTTCGTGCTGGCCGTCGTAATCACATGCTTCTTCGGCTCACTGGTGACCCTTACCTTTGGCGGTGCGTACGTGGTCAAGCTGCTCGAAGAGTACGCCACTGGACCGGCG --referenceAA LDSTFAGLEGVITAVLDEFPHVWAKRRERFVLAVVITCFFGSLVTLTFGGAYVVKLLEEYATGPA --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R2
Aligner Script: Fragment CT_B read 1: python2.7 Fused_read_aligner.py --path CT_fragB_R1/ --infile SERT_CT_DNA1_R1.fast_R1_qc1 --referenceDNA GTTAGTGAGGTTGCCAAAGACGCTGGCCCGAGCCTGTTGTTCATCACATACGCCGAGGCTATCGCGAACATGCCCGCATCTACTTTCTTCGCCATCATCTTTTTCCTGATGCTGATAACCCTTGGGCTGGATAGCACCTTCGCCGGACTGGAGGGTGTCATCACTGCCGTTCTCGACGAGTTCCCCCATGTGTGGGCCAA --referenceAA VSEVAKDAGPSLLFITYAEAIANMPASTFFAIIFFLMLITLGLDSTFAGLEGVITAVLDEFPHVWA --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner Script: Fragment CT_B read 2: python2.7 Fused_read_aligner.py --path CT_fragB_R2/ --infile SERT_CT_DNA1_R2.fast_R2_qc1 --referenceDNA GTTCTGACCGTGGCATTGATCGAGGCCGTGGCCGTAAGCTGGTTTTACGGCATCACGCAGTTCTGTAGGGACGTGAAAGAGATGCTGGGCTTTTCTCCCGGTTGGTTTTGGAGGATTTGTTGGGTAGCAATTAGCCCCCTGTTTCTCCTTTTCATTATTTGCTCTTTTCTGATGTCTCCACCGCAGCTCCGACTGTTTCAGTACAACTACCCC --referenceAA VLTVALIEAVAVSWFYGITQFCRDVKEMLGFSPGWFWRICWVAISPLFLLFIICSFLMSPPQLRLFQYNYP --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R2
Aligner Script: Fragment CT_C read 1: python2.7 Fused_read_aligner.py --path CT_fragC_R1/ --infile SERT_CT_DNA1_R1.fast_R1_qc1 --referenceDNA CTGGATAGCACCTTCGCCGGACTGGAGGGTGTCATCACTGCCGTTCTCGACGAGTTCCCCCATGTGTGGGCCAAGCGACGGGAAAGGTTCGTGCTGGCCGTCGTAATCACATGCTTCTTCGGCTCACTGGTGACCCTTACCTTTGGCGGTGCGTACGTGGTCAAGCTGCTCGAAGAGTACGCCACTGGACCGGCG --referenceAA LDSTFAGLEGVITAVLDEFPHVWAKRRERFVLAVVITCFFGSLVTLTFGGAYVVKLLEEYATGPA --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner Script: Fragment CT_C read 2: python2.7 Fused_read_aligner.py --path CT_fragC_R2/ --infile SERT_CT_DNA1_R2.fast_R2_qc1 --referenceDNA CCCTACTGGTCAATTATCCTGGGCTATTGTATCGGCACCTCAAGTTTCATCTGCATCCCCACATACATCGCATACAGACTGATAATTACGCCCGGTACTTTTAAGGAGCGAATCATTAAAAGCATAACCCCCGAAACCCCCACCGAGATCCCTTGTGGCGATATAAGACTCAATGCGGTC --referenceAA PYWSIILGYCIGTSSFICIPTYIAYRLIITPGTFKERIIKSITPETPTEIPCGDIRLNAV --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R2
MapCounts script: Example: python mapCounts.py --path directory_fragA1_R1/ --infile SERT_NTlibrary_R1.fast_R1_qc1_PRO_qc2
MapRatios script: Example: python mapRatios.py --path ratios_directory/ --templatepath /deepseq_scripts/r_deepseq_scripts/ --infile2 mapcounts_library --infile1 mapcounts_sorted
MapParts script: Example: python mapParts.py --path ratios_directory/ --infile mapratios_sorted_library --mode mutations:1
Unlink script: Example: python mapUnlink.py --path ratios/ --infile mapratios_sorted_library.m1 --type protein --mode ratios --size ##
In Excel, the log(base2) enrichment ratio of the wildtype sequence was subtracted from the log(2) enrichment ratios for all single mutations.
Data was then assembled from the different fragments/reads to span the full sequence in Excel. Where there was overlap, real enrichment ratios were averaged and converted back to log(base2). Maximum depletion was set to log(base2) enrichment ratio = -4.5.
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.
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| |
|
| Submission date |
Jan 23, 2018 |
| Last update date |
Jul 31, 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 |
GPL15520 |
| Series (1) |
| GSE109499 |
Deep Mutagenesis of a Transporter for Uptake of a Non-Native Substrate Identifies Conformationally Dynamic Regions |
|
| Relations |
| BioSample |
SAMN08385402 |
| SRA |
SRX3581238 |
