Total RNA was extracted using hot acidic phenol method (Lin-Chao S and Cohen SN. Cell, 1991. 65(7): 1233-42). Briefly, aliquots (43 mL) of cell cultures grown anaerobically in M9/glucose medium as previously described (Murashko ON and Lin-Chao S. Proc Natl Acad Sci U S A. 2017 Sep 19;114(38): E8025-E8034) were mixed with cold stop solution (5% phenol in ethanol) at an 8:1 ratio. The cells were collected by centrifugation (4000 g, 4°C, 15 min), suspended in 2 mL KJ medium (50 mM glucose, 25 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 100 mM NaCl), then added into boiling 2 mL lysis buffer (0.2 M NaCl, 20 mM Tris-HCl pH 7.5, 40 mM EDTA, 0.5% SDS), and incubated in boiling water for 30 sec followed by addition of 2 mL acidic phenol (pH 4.5). Then, the content of each tube was gently mixed by inverting the tube ~20 times. Total RNA was extracted into aqueous phase by centrifugation (4000 g, 4ºC for 1 h). Then, RNA was precipitated by adding 1 volume of isopropanol and 1/10 volume of 3 M sodium acetate (pH 7.8), and RNA suspensions were stored at -20ºC. Prior use, RNA was precipitated by centrifugation (30,000 g, 4ºC for 15 min), washed with 70% ethanol, centrifugated again (30,000 g, 4ºC for 15 min) and suspended in 20 μL water. The purified RNA was first analyzed by Bioanalyzer (Agilent 2100). The ratio of rRNAs [23S / 16S] greater than 1.4 was considered to correspond to RNA of high quality and therefore it was further used for tiling array.
Label
Cy3
Label protocol
RNA was further converted to labelled cDNA, which was then hybridized with a whole genome tiling arrays (NimbleGen) using standard NimbleGen operating protocols. Briefly, double-stranded cDNA was synthesized using Nimblegen cDNA synthesis kit using 10 μg total RNA and up to 1 μg cDNA was labelled with Cy3-9 mer primers by using Nimblegen labeling kit.
Hybridization protocol
Following labelling, 5 μg of Cy3-cDNA were hybridized for 18 h at 42ºC in MAUIHybridizer with Nimblegen 385K E. coli tilling array by using Nimblegen hybridization kit. Afterwards the array was washed using Nimblegen washing buffers followed by drying on a microarray dryer.
Scan protocol
The microarray slides were scanned using the Axon GenePix 4200A Microarray Scanner at 5 µm resolution and PMT of 480. The scanned images were processed using the NimbleScan software.
Data processing
The array images were further processed using Nimblegen's standard protocol for Nimblescan ChIP data extraction. All raw data were then normalized by using ANAIS (http://anais.versailles.inra.fr/) to obtain gene expression levels.
