Ethachinmate

Approximately 1 g of lichen thalli from each sampling site was washed using autoclaved Milli-Q ultrapure water from Direct-Q UV 5 (Merck Millipore, Burlington, MA, USA). After washing, the lichen thalli were cut into small pieces using flame-sterilized scissors and ground using an autoclaved mortar and pestle. Bulk DNA was extracted from the ground thalli by bead-beating using the ISOIL Large for Beads ver.2 (Nippon Gene, Tokyo, Japan) and precipitated in 70% ethanol with the precipitation-facilitator Ethachinmate (Nippon Gene, Tokyo, Japan) [29 (link)]. The DNA precipitate was resuspended in sterilized ultrapure water, assessed for purity and quantity using a NanoDrop 2000c (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −20 °C until PCR amplification.

We processed all samples after RNA extraction with a DNase kit (Turbo-DNA-free AM1907; Invitrogen, CA, USA) and then ethanol-precipitated the DNA-free RNA samples with Ethachinmate (NIPPON GENE, Japan).
We used the NanoDrop One^C system to determine RNA concentrations and absorption at 260 and 280 nm. We only used samples with a 260/280 ratios ≥2.0. We performed reverse transcription (RT) with 1 µg of RNA using a high-capacity complementary DNA (cDNA) Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA), according to the manufacturer’s protocol.

Total RNA isolated from sorted cells with the use of Isogen and Ethachinmate (Nippon Gene, Tokyo, Japan) was subjected to RT with ReverTra Ace (Toyobo, Tokyo, Japan), and the resulting cDNA was subjected to rtPCR analysis with SYBR Green PCR Master Mix and specific primers in a Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA). Data were normalized by the abundance of β-actin mRNA (Figs 7d and 8e) or attachment region-binding protein (ARBP) mRNA (Supplementary Fig. 1a). The sequences of the various primers (sense and antisense, respectively) were as follows: 5′-AGGTGACAGCATTGCTTCTG-3′ and 5′-GGGAGACCAAAGCCTTCATA-3′ for β-actin, 5′-GGACCCGAGAAGACCTCCTT-3′ and 5′-GCACATCACTCAGAATTTCAATGG-3′ for ARBP, 5′-TCTTCCTCCTGAGGTAATGCTGTCC-3′ and 5′-CACAAAGATCCTGTTTTTGCCAGC-3′ for FBXL5, 5′-GCACCTGAGGCTGACCAATC-3′ and 5′-CATGGGCATACGGTTGTTGAG-3′ for necdin, 5′-GCGCAAACGTCTGAGATGAGT-3′ and 5′-AGAGTTCTTCCATCGTCCGCT-3′ for p57, 5′-CCGGACTATCAGTTGCTAA-3′ and 5′-GGACGTCGCCTGCCTGAA-3′ for HoxB5, 5′-GCTGTCCTCTAAGCGTCACC-3′ and 5′-AGGAGCAGCAGCTCTTCTTG-3′ for MT1, 5′-CAAACCGATCTCTCGTCGAT-3′ and 5′-AGGAGCAGCAGCTTTTCTTG-3′ for MT2, 5′-TGGGTGGAACTGCTCGTAAT-3′ and 5′-AGGATGTAGCGTCCAAATGC-3′ for Slc7a11, 5′-AAGCCGAGAATGCTGAGTTCA-3′ and 5′-GCCGTGTAGATATGGTACAAGGA-3′ for Hmox1 and 5′-CGGCGAGAACGAGAAGAA-3′ and 5′-AAACTTCAGACTCTTTGCTTCG-3′ for Hif1α.

Bulk DNAs were extracted from approximately 10 g of the ground-powdered GM1, GM2, and GM3 samples with the ISOSPIN Soil DNA extraction kit (Nippon Gene Co. Ltd., Tokyo, Japan) and precipitated in 70% ethanol with precipitation facilitator Ethachinmate (Nippon Gene, Tokyo, Japan). The DNA precipitate was resuspended in sterilized ultrapure water. The concentration and purity of the extracted DNAs were checked with NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA) for subsequent procedures and stored at −20 °C. PCR amplicons were generated using the Kapa HiFi HotStart ReadyMix PCR kit (Kapa Biosystems, Wilmington, MA, USA) and the bacterial V3–V4 region-specific primer pair (S-D-Bact-0341-b-S-17, 5′-CCTACGGGNGGCWGCAG-3′/S-D-Bact-0785-a-A-21, 5′-GACTACHVGGGTATCTAATCC-3′) [28 (link)]. The PCR conditions were as follows: 95 °C for 3 min with the lid being heated to 110 °C; 25 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; and a final elongation at 72 °C for 5 min. The sequence library was constructed following our previous method [29 (link)]. Pair-end 300 bp sequencing by MiSeq (Illumina, San Diego, CA, USA) was performed using a Nextera XT Index Kit (Illumina) at Department of Biomedical Science, N-BARD, Hiroshima University.

For northern blot analysis, small RNA (<200 nucleotides) was extracted from mouse liver or lymph using MirVana. Small RNA was condensed with ethachinmate (Nippon Gene), and 2 μg RNA was separated by electrophoresis on 14% polyacrylamide-urea gels and transferred to Hybond-N + membranes (Amersham Biosciences, Uppsala, Sweden). Blots were hybridized with a fluorescein-labeled probe (Gene Images 3′-Oligolabelling Kit, Amersham Biosciences) for the siRNA antisense sequence or mouse U6 sequence. Signals were visualized using a Gene Images CDP-Star Detection Kit (Amersham Biosciences).

exRNA samples were isolated from 5 to 10 mL TCM, lung TCM, liver TCM, and human lung EC-CMs using RNAiso Blood (Takara) with Ethachinmate (Nippon Gene). Total RNA samples were isolated from cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and used to generate cDNA with reverse transcriptase (SuperScript VILO, Invitrogen). For the reverse transcription with GSP, SuperScript III reverse transcriptase (Invitrogen) was used. cDNA from exRNA was amplified using TaqMan PreAmp Master Mix (Applied Biosystems, Foster City, CA, USA) before qPCR analysis. qPCR was performed using SYBR Green Master Mix or TaqMan Fast Advanced Master Mix (Applied Biosystems) in a detection system (StepOnePlus; Applied Biosystems). The gene expression levels were calculated from Ct values, and the relationship between the Ct value and the logarithm of the copy number of the target gene was confirmed to be linear using serial dilutions of the corresponding isolated DNA as a standard. In addition, gene expression levels of nuclear and cytoplasmic samples were normalized to that of Hotair and βactin, respectively. The primer sequences and probes are listed in Supplementary Table S5.