Solexa genome analyzer

Pestalotiopsis fici (W106-1/CGMCC3.15140) was sequenced using a whole-genome shotgun sequencing approach at the Chinese National Human Genome Center (Shanghai, China). Three runs of Roche 454 GS FLX standard pyrosequencing generated 2,999,862 reads (a 24.5-fold sequence depth). The reads were first assembled using Newbler software Version 2.3, which produced 586 contigs. Then a DNA library of 3-kb inserts was constructed and sequenced on an Illumina/Solexa Genome analyzer using a paired-end module to construct the scaffolds. SSPACE and GapFiller software was conducted to further fill the gap and generate scaffolds. The data has been deposited at DDBJ/EMBL/GenBank under accession: ARNU00000000.

We generated small-RNA libraries from the five samples from Yellow River carp using the mirVana^TM mircoRNA Isolation Kit (Ambion, USA), according to Guideline. Five biological replicates were prepared for each sample of three different ovary developmental stages, with each biological replicate comprising RNA extracted from 10 to 50 fish. Five biological replicates were prepared for each sample for both embryo stages, with each biological replicate comprising RNA extracted from 30 to 50 embryos or larvae. A total of 25 small-RNA libraries were prepared from the five samples, with five biological replicates for each sample. Total RNA was ligated with 3’ and 5’ RNA adaptors, and fragments with adaptors on both ends were enriched by PCR after reverse transcription. The subsequent cDNAs were purified and enriched by 6% denaturing polyacrylamide gel electrophoresis to isolate the expected size fractions and eliminate unincorporated primers, primer dimer products, and dimerized adaptors. Finally, the five RNA libraries were sequenced using an Illumina/Solexa Genome Analyzer at Guangzhou Genedenovo Biotech Company (Guangzhou, China).

The fosmid DNA of salt-tolerant clones was sequenced with the Illumina Solexa Genome Analyzer platform. Clean data were obtained by removing the low-quality base at both ends of the raw data and removing the sequences containing the adapter and consisting of a low average base mass, multiple N, and very short length. The sequences were assembled using SPAdes (v3.11.1) to obtain high-quality scaffold fragments. Scaffolds with length > 300 bp were retained for bioinformatics analyses. Megablast against the NCBI plasmid database¹ was adopted to compare and filter the results in line with identity ≥ 30% and math length ≥ 200. cBar (v1.2) and PlasmidFinder (v1.3) were used to identify plasmids and select scaffold fragments as plasmid for genetic prediction. Prodigal (v2.6.3) was used as prediction software. Gene function annotation and secretory protein prediction analysis were carried out using Blast (v2.7.1 plus) and Diamond (v0.9.10) against Gene Ontology (GO), Cluster of Orthologous Groups of Protein (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and nonredundant protein databases (NR). Low-reliability comparison results were filtered out according to identity, e-value, and score values. The optimal results were filtered as functional annotation results for the gene.

ChIP was performed as described above using anti-CHD8 (A301-224A, Bethyl Laboratories). ChIP-DNA was purified and subjected to deep sequencing using the Solexa Genome Analyzer (Illumina). The sequence reads were aligned to the human genome reference (assembly hg19). ChIP-seq peak calling, genomic annotation of peaks and comparison between ChIP-seq and ENCODE datasets were performed using ChIPseeqer (v.2.1) [72 (link)]. An empirical approach was followed to estimate the FDR, which involves using control data set as ChIP-seq data and the ChIP-seq data as the pseudo-control data and running peak detection. The FDR is defined as the ratio of the number of peaks detected in this pseudo-control analysis, to the number of peaks detected in the real ChIP-seq experiment. Motif analysis was performed using FIRE algorithm [73 (link)], included in the ChIPseeqer framework, MEME suite [74 (link)], Weeder PScan [75 (link)] and TRANSFAC database [76 (link)]. CHD8 ChIP-seq data are available from the GEO database (accession number GSE49134). RNAPII, H3K4me3 and PR ChIP-seq data in T47D were previously reported [25 (link)]. GEO accession number for FOXA1 and CTCF ChIP-seq data are GMS803409, GSM803348.

Small-RNA libraries were generated from the nine samples of Yellow River carp: primordial gonad control (PG-CK), primordial gonad exposed to atrazine for 8 h (PG-A8h), primordial gonad exposed to atrazine for 24 h (PG-A24h), juvenile ovary control (IIC-CK), juvenile ovary exposed to atrazine for 8 h (IIC-A8h), juvenile ovary exposed to atrazine for 24 h (IIC-A24h), juvenile testis control (IIX-CK), juvenile testis exposed to atrazine for 8 h (IIX-A8h), juvenile testis exposed to atrazine for 24 h (IIX-A24h). Small-RNA libraries were generated using the mirVanaTM mircoRNA Isolation Kit (Ambion, USA), according to the manufacturer’s instructions. Small-RNA libraries were prepared from three biological replicates for each sample.
Total RNA was ligated with 3′ and 5′ RNA adaptors. Fragments with adaptors on both ends were enriched by PCR after reverse transcription, as described previously [49 (link)]. The resulting cDNAs were purified and enriched with 6% denaturing polyacrylamide gel electrophoresis to isolate the fractions of the expected size and to eliminate unincorporated primers, primer dimer products, and dimerized adaptors [49 (link)]. Finally, the nine resulting RNA libraries were sequenced using an Illumina/Solexa Genome Analyzer, at Guangzhou Genedenovo Biotech Company (Guangzhou, China).

ChIP-DNA was purified and subjected to deep sequencing using the Solexa Genome Analyzer (Illumina, San Diego, CA). The protocol followed to analyze the ChIP-seq data can be found in the Supplementary Methods section.