Nebnext multiplex oligos for kit

The chromatin immunoprecipitation protocol was adapted from previous reports (Buecker et al., 2014 (link)). Briefly, 80–100μL of 48hpf and 72hpf old embryos were crosslinked with 1% formaldehyde for 10min at RT, quenched with glycine at a final concentration of 0.125M, and flash frozen and stored at −80°C. Chromatin was sheared by sonication while emerged in an ice bath to 100–500bp fragments. Sonicated chromatin was incubated with 5–10 μg antibody and incubated overnight at 4 °C. Antibody was bound to Dynabeads by incubating 1mL of antibody bound chromatin with 100μL Dynabead solution at 4°C for 2–4h. ChIP DNA were subject to end repair, A-tailing, adaptor ligation and cleavage with USER enzyme, followed by size selection to 250–500 bp and amplification with NEBNext sequencing primers (Cat#E7335S). Libraries were purified, quantified, multiplexed (with NEBNext Multiplex Oligos for Illumina kit, E7335S). Samples were sequenced with 40-bp single-end read (~150 million reads) on an Illumina HiSEQ2000-seq (BioMicroCenter). Data were processed using the Galaxy sequence analysis platform and aligned to the S. kowalevskii genome using BowTie2 (Goecks et al., 2010 (link); Langmead and Salzberg, 2012 (link); Simakov et al., 2015 (link)). The antibody used for ChIP-seq was H3K27ac from Active Motif (#39133, 39134).

RNA from animal tissue was extracted using the RNeasy Plus Universal Mini Kit (Qiagen), according to the manufacturer’s protocol. RNA from cells was extracted using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from 500ng RNA using the Protoscript II First Strand cDNA synthesis Kit (NEB). Next generation sequencing libraries were prepared as follows. Briefly, 1ul of cDNA prepared above was amplified by PCR with primers that amplify about 150 bp surrounding the sites of interest using KAPA Hifi HotStart PCR Mix (Kapa Biosystems). PCR products were purified (Qiagen PCR Purification Kit/ Gel Extraction Kit) to eliminate byproducts. Libraries were constructed with NEBNext Multiplex Oligos for Illumina kit (NEB). 10 ng of input DNA was amplified with indexing primers. Samples were then pooled and loaded on an Illumina Miseq (150bp single-end run) or Hiseq (100bp paired-end run). Data analysis was performed using CRISPResso (Pinello, L. et al. 2016). A minimum of 100,000 reads were analyzed for all in vivo experiments. RNA-seq libraries were prepared from 300ng of RNA, using the NEBNext Poly(A) mRNA magnetic isolation module and NEBNext Ultra RNA Library Prep Kit for Illumina. Samples were pooled and loaded on an Illumina Hiseq (100bp paired-end run).