Hiseq x10 platform

MO BIO PowerSoil DNA isolation kit provided details about how to manipulate samples; we extracted DNA from soil according to its protocol [51 (link)]. For the stool samples, we extracted metagenomic DNA from approximately 300 mg feces as previously described in the HMP protocol, high temperature was beneficial to the cracking of bacteria, we heated samples at 65 °C for 10 min and 95 °C for 10 min during the operation. Water samples were filtered with sterile 0.22-μm filters, and metagenomic DNA was also extracted from the filter membranes using the MO BIO PowerSoil DNA isolation kit. The DNA was divided into two parts and stored in at − 20 °C until further use. The resulting concentrations and quality of sample DNA were examined via nanodrop instrument and agarose gel electrophoresis.
Based on protocols of NEXTflex Rapid DNA-Seq Kit (Illumina, 96 reactions), some critical process are indispensable to make preparations for sequencing including DNA fragmentation and adapter ligation. We followed the instructions of kit to construct library for sample with the insert size of 350 bp. We lastly sequenced the gut microbiomes of 103 representative samples from 10 bird species and 99 environmental samples (Additional file 10: Table S2). All libraries were then performed sequencing on Illumina Hiseq X10 platform with 2 × 150 bp paired reads (Novogene, Beijing, China).

Genomic DNA libraries with 400 bp insert size were constructed for the four pooled samples of M. normale, one sample each of M. candidum and M. dodecandrum, and 15 of 20 individuals of M. normale sampled from Jianshan using the Illumina TruSeq Library Preparation Kit following the manufacturer’s protocol. These libraries were sequenced on an Illumina Hiseq X10 platform in Berry Genomics, Beijing, and more than 30 Gbp paired end (150-bp) reads were obtained for each library. About 8 Gbp reads were obtained for one sample each of M. candidum, M. normale, and M. dodecandrum. All these short reads were deposited in GenBank with accession numbers SRR8892966-SRR8892971 and SRR19183013-SRR19183030.

Genomic DNA of all 143 isolates was extracted using a Wizard genomic DNA purification kit (Promega, Beijing, China), according to the manufacturer’s instructions. Indexed Illumina sequencing libraries were prepared using a TruSeq DNA PCR-free sample preparation kit (Illumina Inc., San Diego, CA) according to standard protocols. Libraries were sequenced on the Illumina HiSeq X10 platform by 150-bp paired-end strategies according to the manufacturer’s protocols (Bionova, Beijing, China). Raw reads were trimmed by Trimmomatic (55 (link)) and assembled into contigs using SPAdes v3.11.1 (56 (link)). Eight isolates (1120, 1704, 1708, 1709, 1619, 1814, 1217, and ZE5) were further sequenced using the Nanopore platform and assembled using Unicycler under the hybrid assembly mode (57 (link)).

In situ Hi-C was carried out as described^{5 (link)}. Cells were crosslinked with 1% formaldehyde then lysed to collect nuclei. Pelleted nuclei were digested with DpnII restriction enzyme (NEB, R0147). The restriction fragment overhangs were filled and marked with biotin-labelled dATP (Thermo Fisher, 19524016) and dCTP, dTTP, and dGTP before ligation. DNA was reverse crosslinked, purified, and fragmented by sonication on a Covaris sonicator. Biotin-labelled DNA was pulled-down on Streptavidin Dynabeads (NEB, S1420S). After DNA repair and 3′ A addition, SHORT Y-Adaptor (Supplementary Table 1) was added. Diluted DNA on Dynabeads was used for PCR amplification (4, 8, 12,16, and 20 cycles) to produce similar amounts of DNA for sequencing on the Illumina HiSeq X10 platform (paired end 2 × 150 bp reads).

RNA extraction was performed as described previously (54 (link), 58 (link)), with modifications. Cells with or without McdR overexpression (50 ng/mL ATc for 2 h) were harvested and ground in liquid nitrogen. RNA was extracted using TRIzol (Invitrogen) by following the manufacturer’s protocol. qRT-PCR was performed as previously described (54 (link)) using iTaq universal SYBR green supermix (Bio-Rad). The expression level of the sigA gene was used as an internal control. The qRT-PCR data were analyzed by CFX Manager (Bio-Rad). For RNA-seq experiments, rRNA was removed by a Ribo-off rRNA depletion kit (Vazyme). RNA libraries were constructed by using the NEBNext Ultra directional RNA library prep kit for Illumina (NEB). Sequencing was performed on the Illumina HiSeq X 10 platform using 2 × 150-bp paired-end sequencing. FastQC (59 (link)) and Trim Galore were used to trim the raw data. Reads were mapped to M. smegmatis genome (NC_008596) using BWA (60 (link)) and SAMtools (61 (link)). The gene expression levels were analyzed by DESeq2 (62 (link)) in R package (version 3.2.2), and genes were considered differentially expressed at fold change of ≥2 and adjusted P value of <0.05.

Generation of Hi-C libraries with low cell numbers was optimized according to a previous protocol^{11 (link)}. Briefly, 100–600 embryos were cross-linked with 1% formaldehyde for 40 min using vacuum infiltration. Isolated embryo nuclei were digested with 80 U of DpnII (catalog no. R0543L; New England Biolabs) at 37 °C for 5 h. Restriction fragment overhangs were marked with biotin-labeled nucleotides. After labeling, chromatin fragments in proximity were ligated with 4,000 U of T4 DNA ligase for 6 h at 16 °C. Chromatin was reverse-cross-linked, purified and precipitated using ethanol. Biotinylated ligation DNA was sheared to 250–500-base pair (bp) fragments, followed by pull-down with MyOne Streptavidin T1 Dynabeads (catalog no. 65602; Thermo Fisher Scientific). Immobilized DNA fragments were end-repaired, A-tailed and ligated with adapters. Fragments were then amplified with the Q5 High-Fidelity 2X Master Mix (catalog no. M0492L; New England Biolabs). Hi-C libraries were sequenced on the Illumina HiSeq X10 platform (paired-end 2 × 150-bp reads).