Genomic tip columns

For genomic DNA (gDNA) extraction, isolates were grown from single colonies in tryptic soy broth (TSB) liquid cultures overnight at 37°C with shaking at 225 rpm. Cells underwent high-molecular-weight (HMW) DNA extraction using enzymatic lysis with lysozyme, lysostaphin, and proteinase K and Qiagen Genomic-Tip columns, as described previously (64 (link)).
For RNA extraction, cultures grown overnight were diluted, grown to late log phase (optical density [OD] of ∼0.80), and stabilized in RNAlater. Total RNA was isolated and purified by using the Qiagen RNeasy minikit. Lysozyme and lysostaphin were used for cell wall degradation, followed by two cycles of 2 min of bead beating with 1 ml of 0.1-mm silica beads in a mini-bead beater (BioSpec), and RNA was eluted in nuclease-free water. Isolated RNA was treated with 1 μl of Baseline Zero DNase (Epicentre) at 37°C for 30 min, and rRNA depletion was performed by using an Epicenter Ribo-Zero magnetic gold kit (Illumina), according to the manufacturer's instructions.

Specimens of the squid Doryteuthis pealeii were collected by trawl
in the Vineyard Sound by the animal collection department of the Marine Biological
Laboratory in Woods Hole, Massachusetts during the month of July. The giant fiber
lobe (GFL) tissue of the Stellate ganglion, the optic lobe (OL) tissue and a portion
of the sperm sack were manually dissected from a single adult male immersed in
chilled, filtered seawater. The Buccal ganglia (BG), Stellate ganglion with the giant
fiber lobe removed (SG) and Vertical lobe (VL) tissues were dissected from a second
adult male individual. Tissues were also dissected from non-neuronal regions: the
branchial heart, the Gill, the ventral epithelial layer on the pen, the marginal
epithelial layer on the pen, the iridophore layer of the skin, and the chromatophore
layer of the skin. Each of these six tissues originated from a different animal. RNA
from all tissues was extracted with the RNAqueous kit (Life Technologies, Carlsbad,
CA), and genomic DNA was extracted from the sperm sack using Genomic Tip Columns
(Qiagen, Venlo, Limburg, The Netherlands).

A Tn donor plasmid suitable for Tn-seq^{16 (link)} was constructed by amplifying the mariner Tn encoding the Cm-resistance cassette from pAJG39^{29 (link)} using a single 5′-phosphorylated primer PBGSF20, introducing MmeI restriction sites within the inverted repeats of the Tn element. The Tn element was sub-cloned into pJET1.2 (Thermo Scientific) and the resulting plasmid pSV006, was used for in vitro Tn mutagenesis as described in Holt et al.^{71 (link)} with minor adjustments. Briefly, 2 μg of C. jejuni DNA was incubated for 5 h at 30 °C with 1 µg of pSV006 and ~250 ng Himar1-C9 transposase, which was purified as described in Akerley et al.^{72 (link)}. After end-repair with T4 DNA polymerase and E. coli DNA ligase the mutagenesised DNA was transferred to C. jejuni by natural transformation^{13 (link)}. Tn transformants were harvested from plates and pooled. The pooled library was used to inoculate 100–200 ml BHI-TrM-Cm broth to an OD_600nm of ~0.1 and grown overnight), yielding “working stocks”. Genomic DNA was isolated with Genomic-tip columns (Qiagen).