Tissuemiser

RNA was isolated from small pieces (<50 mg wet weight) of frozen breast tumors which were thawed, weighed and immediately homogenized using a Tissue Miser (Fisher), miRNeasy kit followed by DNase treatment, and additional clean-up using RNeasy MinElute Cleanup (all Qiagen). All further steps for RNAseq were conducted by the Mayo Clinic Gene Expression Core, including TruSeq library preparation and Paired-end (PE)150 analysis using HiSeq4000, 8 samples per lane (Illumina), data processing using MapRseq 2.0.0^{30 (link)}, read alignment using TopHat v2 using Mouse Genome Build, mm10 and primary data FastQC. While the initial run yielded reads of up to 150 bases, all sample reads were trimmed to 100 bases for downstream analyses to enhance the quality of the data.

The modified method of amyloid fibers isolation were based on two methods described by Schwartz et al. (2012 (link)) and Romero et al. (2010 (link)). biofilms were grown in eight 10 cm² dishes for 24 h. After washing by PBS for two times, biofilms were scraped and diluted in 1 ml/dish saline extraction buffer (5 mM potassium phosphate, 2 mM MgCl2, 100 mM morpholi nepropane sulphonic acid (Mops) and 1 M NaCl, PH = 7) supplemented with a protease inhibitor mixture. The biofilm suspensions were homogenized using a tissue homogenizer (TissueMiser, Fisher) to shear fibers free from the cell walls. Supernatants were clarified by repeated centrifugation (seven times) at 7000 rpm for 2 min to remove bacteria. At last, supernatants without cells were centrifugation at 16,000g for 20 min to precipitate amyloid fibers, and the precipitate was redissolved in distilled deionized water. Presence of fibers was confirmed via TEM imaging. Amyloid fibers solution was treated by DNase I, RNase and protease K. After treating, amyloid fibers were confirmed by TEM.

Streptococcus pneumoniae (ATCC #6301; serotype 1) were grown on blood agar plates overnight at 37°C and aliquots of 1 x 10⁸ bacteria were recovered as determined by spectrophotometry. The bacteria were washed once in ice-cold PBS and resuspended in PBS at a concentration of 10 x 10⁶ CFU/30 μL. The bacteria were kept on ice until needed. Mice were anesthetized by intraperitoneal injection (100 μL/10g body weight) of xylazine (1mg/mL) and ketamine (7.5 mg/mL) and 30 μL of bacteria culture or sterile PBS was administered to the nares of each mouse. Following infection, the mice were monitored for signs of distress. At 24 and 48h-post infection, the mice were euthanized with CO₂. The lungs were removed and homogenized for 30 sec in sterile PBS (1 mL) using a tissuemiser (Fisher Scientific). For the survival experiments, WT and NKLAM-KO mice were infected nasally with 5 x 10⁷S. pneumoniae CFU and monitored several times a day for signs of severe sickness (e.g. lethargy, hypothermia, piloerection, inability to maintain upright posture). The total duration of the experiment was 120 hours. Extremely ill mice were euthanized immediately by CO₂ asphyxiation and scored dead for the purposes of the experiment.

Brain and ceca tissue samples were homogenized in Trizol reagent (Invitrogen, Carlsbad, CA, USA) using a hand-held Tissuemiser (Fisher Scientific, Hampton, NH, USA) and total RNA was extracted according to the manufacturer’s instructions. Tissue samples were collected for expression analysis from two individuals at each of nine time points over a 48-h period (6-h intervals), for each photoperiod treatment. 100 ng of total RNA were used to generate cDNA using the SuperScript VILO MasterMix RT-PCR kit (Invitrogen, Carlsbad, CA, USA). RealTime PCR was performed using gene-specific primers (Integrated DNA Technologies, Coralville, IA, USA) and PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) on a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), and using Actin as the housekeeping gene. PCR conditions were 50 °C for 2 min, 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 57 °C for 1 min. The primers used for qPCR of clock genes were the same as reported in Okano et al. (2001) (link). Primer sequences are shown in Table S1.