Truseq v3 cluster kits

Whole genome bisulfite sequencing libraries were prepared from 1 µg of DNA following Illumina’s “Whole- Genome Bisulfite sequencing for Methylation Analysis” protocol. Three lanes of paired end 100 bp sequencing was performed for each of the library on the Illumina HiSeq2500 platform using the TruSeq v3 cluster kits and SBS kits to achieve coverage ranging between 25–30x. (details in Supplementary Table S1). Supporting DNA methylome data on the core and two healthy weight male subjects was obtained using Illumina Infinium Bead Chip Human450 arrays. Further experimental details, details of quality assessment and sequence alignment, and analysis of differential methylation and methylation profiles are provided in Supplementary Information and Supplementary Table S1.

WGBS libraries were prepared following Illumina’s “Whole-Genome Bisulfite Sequencing for Methylation Analysis” protocol. Briefly, 1 μg of genomic DNA was spiked with 0.5% unmethylated lambda DNA and sonicated to generate fragments of size between 150 to 300 bp. Library preparation was performed using Illumina’s Paired-end DNA Sample Prep Kit (discontinued, Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. The size-selected libraries were then subjected to bisulfite conversion as previously described [55 (link)]. Adaptor-ligated bisulfite-treated DNA was enriched by 10 cycles of PCR amplification using the PfuTurbo Cx Hotstart DNA Polymerase (Stratagene, La Jolla, CA, USA). Qualitative and quantitative checks of the libraries were performed using Agilent’s High sensitivity DNA kit (Agilent, Santa Clara, CA, USA) and KAPA Library quantification kit (KAPA Biosystems, Wilmington, MA, USA). Three lanes of paired-end 100 bp sequencing was performed for each of the libraries on the Illumina HiSeq2500 platform using the TruSeq v3 cluster kits and SBS kits (Illumina) to achieve coverage ranging between 25 × and 30 ×.

For two individuals, RNA was extracted from the entirety of each specimen for two “whole body” RNA sequencing libraries. For the other individual, select tissues were microdissected (silk glands, venom glands, and brain tissue), and used for 6 tissue-specific RNA sequencing libraries (S1 and S2 Tables). In all cases, RNA was extracted using a combined TRIzol (Ambion, Life Technologies) plus column-based protocol. Each of the prepared RNA samples were treated with TURBO-free DNase (Life Technologies), and ribosomal RNA content was depleted with the Ribo-Zero Gold Kit (Epicentre, Human/Mouse/Rat). Strand-specific RNA sequencing libraries were constructed using the NEBnext Ultra Directional RNA Library Prep Kit (NEB, protocol “B”) and barcoded using TruSeq RNA adapters (Illumina). All C. darwini RNA sequencing libraries are provide (S2 Table). High-throughput RNA sequencing was performed on the Illumina HiSeq2000 (100 x 100) platform using TruSeq v3 cluster kits and TruSeq SBS chemistry (Illumina).