Serratia symbiotica CWBI Tn-seq experiment pool
DNA was isolated from cells using a PureLink Genomic DNA Mini Kit (Invitrogen) and sheared using a Covaris S220 to an average size of 400 bp. Poly-C tails were added to the fragmented DNA using terminal deoxynucleotidyl transferase (Promega). Then, the processed fragments were size-selected using AMPure XP beads (Beckman Coulter) and amplified by PCR with a biotinylated primer binding inside of the transposon and a non-biotinylated primer binding to the poly-C tail that also included an internal six-base barcode. Amplified library fragments were then bound to streptavidin M-280 Dynabeads (ThermoFisher) and washed to remove unbound DNA. Illumina adapters with external barcodes were added in a subsequent PCR reaction that also introduced an internal six-base dual barcode in the other read so that the resulting DNA fragment library had dual internal barcode combinations unique to each sample and the same external barcodes. Each sample was run individually on a BioAnalyzer 2100 (Agilent) to check its fragment size distribution. Then, all samples were pooled and sequenced on a NovaSeq 6000.   Internal barcode pairs comprising bases 2-7 of read 1 and bases 1-6 of read two were as follows for each sample (R1/R2): library_replicate_1 (TGAAGG/CCTTCA), library_replicate_2 (AACATA/CCTTCA), library_replicate_3 (GATACA/CCTTCA), culture_replicate_1_passage_1 (TGAAGG/GACGCG), culture_replicate_1_passage_2 (AACATA/GACGCG), culture_replicate_1_passage_3 (GATACA/GACGCG), culture_replicate_1_passage_4 (GGTGTG/GACGCG), culture_replicate_2_passage_1 (TGAAGG/TGTATC), culture_replicate_2_passage_2  (AACATA/TGTATC), culture_replicate_2_passage_3  (GATACA/TGTATC), culture_replicate_2_passage_4  (GGTGTG/TGTATC), culture_replicate_3_passage_1 (TGAAGG/CACACC), culture_replicate_3_passage_2  (AACATA/CACACC), culture_replicate_3_passage_3  (GATACA/CACACC), culture_replicate_3_passage_4  (GGTGTG/CACACC), aphid_replicate_1 (TGAAGG/TATGTT) aphid_replicate_2 (AACATA/TATGTT), aphid_replicate_3 (GATACA/TATGTT). Reads were demultiplexed from the pooled sample allowing one mismatch to each internal barcode.