This readme.txt file was generated on 2023-01-19 by Alisha Block. Recommended citation for the data: Tischler, Anna D; Block, Alisha; Palani, Nagendra; Brokaw, Alyssa; Zhang, Leanne; Namugenyi, Sarah; Beckman, Kenneth. (2022). Mycobacterium tuberculosis Erdman ATCC 35801 arrayed Himar1 transposon library isolated using MtbYM rich medium. Retrieved from the Data Repository for the University of Minnesota. https://doi.org/10.13020/wt3q-0405. ------------------- GENERAL INFORMATION ------------------- 1. Title of Dataset: Mycobacterium tuberculosis Erdman ATCC 35801 arrayed Himar1 transposon library isolated using MtbYM rich medium. 2. Author Information Author Contact: Anna D Tischler (tischler@umn.edu) Name: Anna D Tischler Email: tischler@umn.edu Name: Alisha Block Email: bloc0078@umn.edu Name: Nagendra Palani Name: Alyssa Brokaw Name: Leanne Zhang Name: Sarah Namugenyi Name: Kenneth Beckman 3. Date published or finalized for release: 2022-08-18 4. Date of data collection (single date, range, approximate date): 2017-04-21 to 2018-07-20 5. Geographic location of data collection (where was data collected?): NA 6. Information about funding sources that supported the collection of the data: This work was supported by a National Institutes of Health Director's New Innovator Award DP2AI112245 from NIAID (ADT), T90 DE02273 from NIDCR (AMB) and a University of Minnesota Doctoral Dissertation Fellowship (SNB). 7. Overview of the data (abstract): We created a Himar1 transposon library in Mycobacterium tuberculosis Erdman ATCC 35801 strain containing ~8000 mutants arrayed in 80 tube racks in a 96-well format. The transposon library was isolated on MtbYM rich medium that allowed recovery of auxotrophic mutants that would not be viable on standard Middlebrook M. tuberculosis culture medium. Using orthogonal pooling and transposon sequencing (Tn-seq), we mapped the location of Tn mutants in the library. The library was split into two groups of 40 racks. For each set of 40 racks, pools were created of all mutants in each row (rows A-H), column (columns 1-12) and rack (racks 1-40 or 41-80) to generate 60 Tn-seq samples for Illumina sequencing. Two different mapping methods, the heuristic Straight Three strategy and the probabilistic Knockout Sudoku algorithm, were used to map the arrayed library with good agreement between both mapping methods. This repository contains Illumina sequencing data for all rack, column, and row pools used to map the entire 80 rack arrayed library. -------------------------- SHARING/ACCESS INFORMATION -------------------------- 1. Licenses/restrictions placed on the data: CC0 1.0 Universal (http://creativecommons.org/publicdomain/zero/1.0/) 2. Links to publications that cite or use the data: Block, Alisha M., Namugenyi, Sarah B., Palani, Nagendra P., Brokaw, Alyssa M., Zhang, Leanne, Beckman, Kenneth B., Tischler, Anna D. 2023. Mycobacterium tuberculosis Requires the Outer Membrane Lipid Phthiocerol Dimycocerosate for Starvation-Induced Antibiotic Tolerance. mSystems 8(1). https://doi.org/10.1128/msystems.00699-22 3. Was data derived from another source? No If yes, list source(s): 4. Terms of Use: Data Repository for the U of Minnesota (DRUM) By using these files, users agree to the Terms of Use. https://conservancy.umn.edu/pages/drum/policies/#terms-of-use --------------------- DATA & FILE OVERVIEW --------------------- File List Filename: 1-40.zip Short description: Illumina sequencing files for transposon library mapping racks 1-40 Filename: 41-80.zip Short description: Illumina sequencing files for transposon library mapping racks 41-80 2. Relationship between files and description: The transposon library was sequenced in groups of 40 racks (1-40 and 41-80). All Illumina sequencing files for each group are combined into one file. File 1-40.zip contains all the reads for the column, row, and rack pools for racks 1-40. File 41-80.zip contains all the reads for the column, row, and rack pools for racks 41-80. Each rack is in a 96-well format with columns A-H and rows 1-12. Each rack is numbered 1-80. File names: Col = column pool Plate = rack pool Row = row pool Each sample has two Illumina fastq files, “R1” and “R2”, corresponding to the paired-end reads for the sample. -------------------------- METHODOLOGICAL INFORMATION -------------------------- 1. Description of methods used for collection/generation of data: Transposon (Tn) mutagenesis of wild-type M. tuberculosis Erdman was performed by transduction with the mycobacteriophage phAE159 carrying the Himar1 Tn as previously described. Wild-type bacteria were grown to mid-exponential phase (OD600 of 0.4-0.6) in 7H9 broth, washed and resuspended in MP buffer (50 mM Tris pH 7.6, 150 mM NaCl, 10 mM MgCl2, 2mM CaCl2), then transduced with mycobacteriophage at a MOI of at least 20:1 for 3 hours at 40˚C. Phage adsorption was stopped with Stop Buffer (MP buffer with 60 mM Na-citrate and 0.6% Tween-80) and transduced cells were plated on MtbYM agar pH 6.6 with Kan at a density of 100-200 colonies per plate. Plates were incubated at 37˚C with 5% CO2 for at least 3 weeks. Approximately 8000 individual Tn mutant colonies were picked from plates into 600 ul of MtbYM broth in 1 ml v-bottom Matrix screw-cap tubes in a 96-well rack (Thermo Scientific) and incubated with shaking at 37˚C for 2 weeks, until turbid. Tn mutants were orthogonally pooled using the Straight Three strategy and sequenced, as previously described. Briefly, the Tn library was pooled in 2 groups of 40 racks each (racks 1-40 and 41-80). For each rack, a small volume of culture was removed from each tube and combined appropriately to form 8 row pools (rows A-H), 12 column pools (columns 1-12) and a rack pool. Each individual rack pool was aliquoted into one sample for sequencing and nine 1 ml aliquots for experimental use, which were stored at -80˚C with glycerol at 15% final concentration. After pooling, glycerol was added at 15% final concentration to each Tn mutant culture and racks were stored at -80˚C. For each group of 40 racks, analogous row and column pools were pooled from all 40 plates to generate 8 row and 12 column samples. These were multiplexed with the 40 individual rack pools for a total of 60 samples for Tn-seq sequencing. Genomic DNA (gDNA) was extracted from each row, column and rack pool using the CTAB-lysozyme method and submitted to the University of Minnesota Genomics Center (UMGC) for library creation and Tn-seq sequencing as previously described. Briefly, M. tuberculosis genomic DNA was fragmented with a Covaris S220 ultrasonicator and a whole genome library was prepared using the TruSeq Nano library preparation kit (Illumina). Library fragments containing Tn junctions were PCR-amplified from the whole genome library using the Tn-specific primer Mariner_1R_TnSeq_noMm and Illumina p7 primer (Table S6). The amplified products were uniquely indexed to allow sample pooling and multiplexed sequencing. Resulting Tn-seq libraries were sequenced on an Illumina 2500 High-output instrument in 125-bp paired-end output mode using v4 chemistry (Illumina). 2. Methods for processing the data: Tn mutants associated with the reads were traced back to their rack location in the arrayed Tn library using two approaches: Straight Three and Knockout Sudoku. Reference for Straight Three mapping: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6134198/pdf/mSystems.00062-18.pdf Reference for Knockout Sudoku mapping: https://www.nature.com/articles/ncomms13270