normalization data transformation protocol
We used two original programs (kSusageA, kSusageB) witten in objective-C to analyze the Super SAGE data. The FASTAQ format fileswere split every 12,000,000 lines using Terminal. These files were processed to create FASTA format files using kSusageA. The kSusageA program extracts 26-base tags from reads of 35 bases, sorts the tags based on index sequence, and counts the frequency of the tags. The tags were compared to the NCBI mouse transcript database using BLAST. The reference sequence DNA IDs were picked from the results of BLAST analysis, and the tags were annotated using kSusageB. Supplementary_files_format_and_content: the frequency of the tags
Male C57BL/6 mice that were 4-weeks old (Charles River Laboratories, Tokyo, Japan) were housed in a temperature-controlled environment (23 ± 1°C) with a 12 h light/dark cycle.
sample treatment protocol
The animals were randomly divided into four groups: normal diet (D12450B: Research Diets, Inc., New Brunswick, NJ, USA), normal diet with 1% WEPAK, high-fat diet (D12451: Research Diets, Inc.), and high-fat diet with 1% WEPAK (4 mice / group).
nucleic acid library construction protocol
Total RNA was extracted from liver samples with an RNeasy lipid tissue kit. Double-stranded cDNA was synthesized with biotinated oligo-dT primer and the Double-Stranded cDNA Synthesis kit (Invitrogen) from 5 µg of total RNA. Double-stranded cDNAs were digested with NlaIII (New England Biolabs, Beverly, MA, USA) after being purified using a PCR purification kit (QIAGEN). Double-stranded cDNAs digested with NlaIII were bound to Dynabeads M270 streptavidin (Invitrogen). Adapter sequence 2 containing an EcoP15I restriction site was ligated to the double-stranded cDNAs that were bound to Dynabeads. Double-stranded cDNAs were digested with EcoP15I (New England Biolabs). Double-stranded cDNAs digested with EcoP15I and separated from Dynabeads were tag sequences. Adaptor sequence 1 that contained index sequence was ligated to tag sequences. Tag sequences were then amplified by PCR and gel purified.