Mirneasy mini kit protocol

Total miRNA was isolated from purified primary cerebrovascular endothelial cells (pCECs) using the miRNeasy mini kit protocol (Qiagen, Valencia, CA), and then converted to cDNA using oligo dT, random hexamers with Superscript RT II enzyme (Invitrogen, Grand Island, NY). Real-time PCR was performed using SYBR Green master mix (Qiagen, Valencia, CA) and predesigned primers (synthesized by Qiagen), normalized to internal control miR-39 expression.

After collection, lungs were immediately placed in RNA Later (Sigma Aldrich, USA) and stored at − 20 °C. Samples were homogenized in QIAzol® Lysis Reagent. Total RNA was extracted with the miRNeasy Mini Kit protocol (QIAGEN, Germany), using an automated method (QIAcube: QIAGEN, Germany) adapted to include DNase I treatment. RNA concentration was measured using Qubit 4 fluorometer (Thermo Fisher Scientific, USA). RNA integrity was assessed by checking the 2:1 ratio of 18S and 28S ribosomal RNA bands and RNA Integrity Number (RIN) by agarose gel electrophoresis and Bioanalyzer RNA 6000 Nano Kit (Agilent, USA), respectively. Lung-derived RNA was suitable for RNA-sequencing (RIN > 8). Libraries for high-throughput RNA sequencing were prepared using the QuantSeq FWD kit (Lexogen, Austria) and sequenced in three different runs with the Illumina NexSeq500 platform, which generated at least 20 million reads for each sample. 96% of the reads were mapped to the rabbit genome.

The severity of DSS-induced injuries increases from the proximal to the distal colon in C57BL/6 mice. Consequently, we focused our analysis on this section of the intestine. RNA was extracted from distal colon tissues by using the miRNeasy mini kit protocol (Qiagen, Hilden, Germany) and further processed by using the IVT PLUS protocol (Affymetrix, Santa Clara, CA, USA). All processed RNA samples were hybridized on Affymetrix GeneChip® Mouse Genome 430 2.0 Arrays. CEL files were processed by using software packages from the Bioconductor suite of microarray analysis tools for the R statistical software environment [78 ]. Arrays that passed the quality controls (QC) were background corrected and normalized by the frozen robust multiarray method to generate the expression values [79 (link)]. The QC metrics—obtained using the affyPLM package—examined the distribution of log intensities, normalized unscaled standard error, relative log expression, and median absolute value relative log expression as well as the general aspect of the array pseudo- and raw images [80 ]. Additionally, the Mouse4302_Mm_ENTREZG v16.0 Brainarray Custom CDF environment was used for probe set-level summary to obtain the expression data matrix [81 (link)]. The array data have been deposited in the ArrayExpress public repository (E-MTAB-8543).