Nanodrop 1000

Total cellular RNA was extracted from 1× 10⁵ Lin⁻CD45⁻Sca-1⁺ CD31⁻ cells from younger or older mice heart, for three samples, isolated using FACS, 10 mice as a pool for per sample. RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. RNA concentrations were determined photometrically using a NanoDrop 1000 (Peqlab, Erlangen, Germany). Overall RNA quality was assessed by 1% agarose gels with 1 kb molecular weight marker separated in parallel.

We extracted mRNA with Trizol isolation using Qiazol Lysis Reagent and a bead mill. RNA had to be concentrated by vacuum centrifugation and concentrations were measured by spectrophotometry with NanoDrop 1000 (PeqLab, Germany). RNA integrity was tested with automated electrophoresis with Experion Automated Electrophoresis System (Bio-Rad Laboratories, USA). Next, RNA was transcribed into cDNA with RevertAid First Strand cDNA Synthesis Kit (Fermentas, USA). PCR reactions were performed on Rotor-Gene Q (QIAgen, Germany) in a total reaction volume of 20 μL with Brilliant II SYBR Green qPCR Master Mix (Stratagene, USA) and forward and reverse primers (Biomers, Germany). Expression analyses were performed as reported^{31 (link)}. Primer sequences and complete methods are given in the supplemental section (supplementary methods and Suppl. Table 1).

RNA was isolated using RNeasy Kit (Qiagen). RNA was purified and washed via precipitation with 80% ethanol and quantified using NanoDrop 1000 (Peqlab). For reverse transcription and NGS library preparation, the NEBNext Ultra RNA Library Prep Kit for Illumina (E7530) and NEBNext Multiplex Oligos for Illumina, Dual Index Primer Set 1 (E7600) were used according to the manufacturer’s instructions. DNA fragments and index PCR products were purified with HighPrep (MagBio) beads. The sequencing library concentration was determined using the Quant-iT PicoGreen dsDNA Assay-Kit (Thermo).

For molecular analysis, intestinal tissue samples were snap frozen in liquid nitrogen and kept at −80 °C until further processing. After tissue collection in cryo-tubes containing 1.4 mm ceramic beads and homogenization in a FastPrep (Precelly 24, Peqlab, Erlangen, Germany) at 5000× g for 15 s, RNA was isolated by phenol-chloroform extraction using peqGold RNA TriFast (PeqLab, Erlangen, Germany), as previously described [45 (link)]. The purity of the obtained RNA was measured using 260/280 ratios of optical densities (Nanodrop 1000, PeqLab, Erlangen, Germany). Complementary DNA was obtained by reverse transcription using M-MLV transcriptase and random hexanucleotide primers (Invitrogen, Carlsbad, CA, USA). Relative mRNA expression levels were analyzed with quantitative real-time PCR (Bio-Rad, Feldkirchen, Germany). The ratio between the gene of interest (Occludin, sense: 5′-CCCAGGTGGCAGGTAGATTA, antisense: 5′-AGGCCTGTTTTGTGAGCACT; Claudin-1, sense: 5′-GTGGTGTTGGGTAAGAGGTTGT, antisense: 5′-AAAAGATGTGGATGGCTGTCA; Zonulin, sense: 5′-ACTGGGTCCAGGAAACAATG, antisense: 5′-TCCTCTTCCAGGGTGAATTG) and the reference gene (Cyclophilin A, sense: 5′-GGCAAATGCTGGACCAAACAC, antisense: 5′-TTAGAGTTGTCCACAGTCGGG AGATG) was calculated and expressed as the fold change relative to the control group.

Genomic DNA was isolated from buffy coats using a ReliaPrep™ Blood gDNA Miniprep System (Promega, A5082) according to manufacturer’s instructions. 200 µl of sample were mixed with cell lysis buffer and proteinase K and incubated at 56 °C for 10 min. Lysates were transferred onto binding columns and centrifuged for 1 min at 14,000×g. Columns were washed three times with provided washing solution. Then, membrane-bound DNA was eluted in 50 µl nuclease-free water. Concentration and quality of DNA were assessed using a NanoDrop 1000 spectrophotometer (ND1000, PeqLab).