Extraction of the whole genome and total RNA
Single colony was picked up from XM solid medium and inoculated into TSB
liquid medium (Sigma, Germany) in flasks. 5 mL cell samples were
collected after the shaking incubation at 34 °C for
48~72 h and were used for genome extraction. 400 to 600
μL TSB culture was centrifuged at 12000 rpm for 3 min for pellet
collection. Pellets were then washed by 600 μL 0.05 M
N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid sodium salt
(TES) buffer (pH=8.0) twice and resuspended in 700 μL TES buffer. 50
mg/mL of lysozyme and 4 mg/mL RNase were sequentially added to a final
concentration of 5 mg/mL or 40 μg/mL, respectively. Pellets with enzymes
were incubated at
37
°C for at least 1 h. The tubes should be gently inverted 4 to 5 times
during incubation until the liquid became sticky. 10% sodium dodecyl
sulfate was added to a final concentration of 1~2%. 5
μL of 20 mg/mL protease K was also needed for protein denaturation. The
mixture was then incubated at 37 °C for 30 min, during which the tubes
were vigorously inverted 2 to 3 times until the liquid was transparent.
To precipitate the protein, 100 μL of 5 M NaCl and 50 μL of 10% CTAB
preheated to 65 °C were added in tubes. The mixture was then incubated
at 65 °C for 1 h. The tubes were inverted each 15 min. To extract DNA
from the liquid, equivalent volume of phenol:chloroform:isoamyl alcohol
(25:24:1) saturated with 10 mM Tris, pH 8.0, 1 mM EDTA was used and then
vibrated in vortex for 15 s. The mixture was centrifuged at 12000 rpm
for 10 min. Clear supernatant was transferred into new tubes and
extraction by phenol:chloroform:isoamyl alcohol (25:24:1) was repeated 3
times. To precipitate DNA, 3 M NaAc was added to a final concentration
of 0.3 M. 4 °C isopropanol was added before DNA samples were kept in -20
°C overnight. The mixture was then centrifuged at 12000 rpm for 1 min at
4 °C and the supernatant was discarded. 2 mL cold ethanol was used to
wash samples and the supernatant was discarded after 12000 rpm
centrifugation for 10 min. 1 mL cold 70% ethanol was then added and
supernatant was also discarded. At last, 30 to 50 μL TE buffer preheated
to 55 °C was added in tubes to dissolve genome. Concentration of genome
was measured by NanoDrop spectro-photometer. Genome sequencing was
performed on Illumina Hiseq2000 platform (BGI, China).
For RNA isolation, two replicates from independent cultures in modified
minimal liquid cultures were used. Cells were harvested at two time
points, i.e. 10 h in the early exponential phase and 50 h at the onset
point of erythromycin biosynthesis, for total RNA extraction. 10 mL
culture was centrifuged at 4000×g at 4 °C for 10 min. The broth
supernatant was discarded and the cells were resuspended in 2 mL
RNAlaterTM solution (Invitrogen, USA). Total RNA was
extracted with an
RNeasy
Plus Mini kit (Qiagen, Germany) using glass beads to mechanically
disrupting cells with a FastPrep (30 s ×4; M.P. Biomedical, USA). DNase
treatment by RNase-Free DNase Set (Qiagen, Germany) aided to digest DNA
in samples. The RNA integrality was analyzed by 1% agarose gel
electrophoresis and Bioanalyzer (Agilent, USA). The RNA quality was also
determined by Bioanalyzer (Agilent, USA). 1 μg total RNA was reverse
transcribed using a PrimeScriptTM RT Reagent Kit with
gDNA Eraser (Takara, Japan) for RT-qPCR. RNA sequencing was accomplished
using BGIseq 500 next-gen sequencer or Illumina Hiseq4000 sequencer,
which was commercially provided by BGI, China or Shanghai Majorbio
Bio-pharm Technology Co.,Ltd, respectively. The original image data
obtained from sequencers was transferred into sequence data via base
calling, which is defined as raw data or raw reads and saved as FASTQ
file. Datasets consisted of at least 30 M pair-ended reads per sample
with a 100- or 150-bp read length. After sequencing, the raw reads were
filtered includes removing adaptor sequences, contamination and
low-quality reads from raw reads. Especially for genome raw reads, reads
with a large amount of duplications or N base were also removed.