Le220 system

At 72 hpf, heads were dissected from ICH+ larvae treated for 24 h with ramipril, quinapril or a DMSO control (n=5). Samples were lysed in S-Trap buffer and sonicated using a Covaris LE220+ system at 500 W for 8 min. Proteins were reduced, alkylated and digested using the standard S-Trap column protocol and trypsin digestion enzyme at 2 µg/µl. Peptides were eluted into a solution of 30% aqueous acetonitrile with 0.1% formic acid. Peptides were desalted using POROS R3 beads and eluted into a final solution of 30% aqueous acetonitrile with 0.1% formic acid. Samples were completely dried in a Heto speed vacuum centrifuge.

DNA from samples 3-4D, 5-3D, 6-6B, 5-2D, 5-4B, 2-2B, 2-3B, 2-4A, 3-3A and 3-4A was prepared with TruSeq Nano DNA Library Prep Kit (Illumina) according to the manufacturer's protocol: 100 ng of genomic DNA were fragmented to 350 bp using Covaris LE220 system (Covaris, Inc.). Fragments were end-repaired, A-tailed, adaptor ligated and PCR amplified (8 cycles). The final libraries were validated using Agilent Tapestation 2200 (Agilent Technologies) and Qubit flourometer (Invitrogen), normalized and pooled in equimolar ratios. 101 bp paired-end sequencing was performed on the Illumina HiSeq 4000 according to the manufacturer's protocol. To match downstream analysis requirements, only the first members of the read pairs were considered, and were truncated to the first 50 bp. The resulting genome coverage was between 0.25 and 0.39 × (0.64–0.79 × for paired end reads). Data analysis were performed following Scheinin et al.49 (link)