Illumina HiSeq 2500 paired end sequencing; Insertional mutagenesis in the zoonotic pathogen Chlamydia caviae
Insertional mutagenesis in the zoonotic pathogen Chlamydia caviae
Bacterial genomic DNA was isolated from infected Vero cell cultures. In brief, bacteria were harvested from infected cells at 32 hpi by H2O-mediated lysis and sonication. Remaining intact cells and host cell nuclei were removed by centrifugation at low speed (1600 rpm, 10 min). Released bacteria were collected by centrifugation at high speed (17,000 x g, 15 min), resuspended in SPG buffer, and treated for 1 h at 37°C with DNase I (NEB) to remove host DNA. Bacteria were then washed once with PBS, resuspended in a small volume of PBS, followed by heat-inactivation of residual DNase I at 75°C for 20 min. Expected insert sequence incA::GII: GTGAAGTAGGGAGGTACCGCCTTGTTCACATTACTGTGACTGGTTTGCACCACCCTCTTCGGGAACCGTACGTACCCCTCTCGGAGTATACGGCTCTGTTATTGTTCGTTCGTAAAAATTCACTGTCGACATTCACTTGTGTTTATGAATCACGTGACGATGACAATGAAAGCATACAACAAGAGTTTTACGTTGTTTCGCTATCATTGCCATTTCCCAACGCGTGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCATATGCGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAGACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATGCATGCACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTACGCGTCGCCACGTAATAAATATCTGGACGTAAAAGATAACGATAAAGTCTTGTAAAAACTTCGTCTATATTTTCTTGTGAATTTTTACTGATTCTTTCTAAAATTGCCATTGTTGGTTTCATTTTGAGGTTTTCCTCCCTAATCAATTTTTAATTTTAGTACACAATAACTGTACCCCTTTGCCATGTAAAGGGCGTTACCCTTCTCAGACTACTACGAGTACTCCGTACCCTTGCAAGATTTTCAAGCTCTAGTGCTATAGCCTTTTTCCTCCTTTCTATTAGGCATTCTTGTTTAGGGTATCCCCAGTTAGTGTTAAGTCTTGGTAAATTCAGATTCTCGGCATCGCTTTCGTTTCGTTCCCATAGGTTCTCCTACAGATTGTACAAATGTGGTGATAACAGATAAGTCGTGTTGTCTAACTTACCTTTCTTTGTACTAGAGGTTTCAGACACTTTCCTCTATCGAGGACTAATCGAAATTAGAAACTTGCGTTCAGTAAACACAACTTATACCTTATATCTGATTAACATTGCGACTCAGTCGTACCCGATTGTCTTTAGGTAACTCATCGCTTTCCAACCGTGCTCTGTTCCCGTATCAGCTTTCGCTTTTCGGTTAGGTTGGCTGTTTTCTGTGTTATCTTACAGAGTAGTACCTTAAACTACTTGACTTAACACCCTATCTGGGCGCAC; sinC::GII: GTGAAGTAGGGAGGTACCGCCTTGTTCACATTACTGTGACTGGTTTGCACCACCCTCTTCGGGAACCGTACGTACCCCTCTCGGAGTATACGGCTCTGTTATTGTTCGTTCGTAAAAATTCACTGTCGACATTCACTTGTGTTTATGAATCACGTGACGATGACAATGAAAGCATACAACAAGAGTTTTACGTTGTTTCGCTATCATTGCCATTTCCCAACGCGTGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCATATGCGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAGACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATGCATGCACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTACGCGTCGCCACGTAATAAATATCTGGACGTAAAAGATAACGATAAAGTCTTGTAAAAACTTCGTCTATATTTTCTTGTGAATTTTTACTGATTCTTTCTAAAATTGCCATTGTTGGTTTCATTTTGAGGTTTTCCTCCCTAATCAATTTTTAATTTTAGTACACAATAACTGTACCCCTTTGCCATGTAAAGGGCGTTACCCTTCTCAGACTACTACGAGTACTCCGTACCCTTGCAAGATTTTCAAGCTCTAGTGCTATAGCCTTTTTCCTCCTTTCTATTAGGCATTCTTGTTTAGGGTATCCCCAGTTAGTGTTAAGTCTTGGTAAATTCAGATTCTCGGCATCGCTTTCGTTTCGTTCCCATAGGTTCTCCTACAGATTGTACAAATGTGGTGATAACAGATAAGTCCTAGAGCTTAACTTACCTTTCTTTGTACTAGAGGTTTCAGACACTTTCCTCTATCGACTATCAACCGAAATTAGAAACTTGCGTTCAGTAAACACAACTTATACCTTATATCTGATTAACATTGCGACTCAGTCGTACCCGATTGTCTTTAGGTAACTCATCGCTTTCCAACCGTGCTCTGTTCCCGTATCAGCTTTCGCTTTTCGGTTAGGTTGGCTGTTTTCTGTGTTATCTTACAGAGTAGTACCTTAAACTACTTGACTTAACACCCTATCTGGGCGCAC Bacterial genomic DNA was isolated using the DNeasy Blood and Tissue kit (Qiagen) and was quantified using the Qubit dsDNA BR Assay kit (Thermo Fisher Scientific). The genomic DNA of each sample was randomly sheared into short fragments of about 350 bp respectively. The obtained fragments were subjected to library construction using the NEBNext® DNA Library Prep Kit, following the instructions strictly. Briefly, as followed by end repairing, dA-tailing, and further ligation with NEBNext adapter, the required fragments (in 300-500 bp size) were PCR enriched by P5 and indexed P7 oligos. After purification and subsequent quality check, the library is ready for sequencing.
Experimental Factor: genotype
sinC::GII