Rneasy microkit columns

Blocks of 2–4 cm were taken from the placental parenchyma, briefly washed in phosphate buffer saline, snap frozen in liquid nitrogen, and stored in − 80 °C until homogenization. Half of the biopsy was homogenized in TRIzol reagent (Invitrogen, Life Technologies) on ice with a tissue grinder (Sigma Aldrich). Total RNA was extracted using TRIzol reagent (Invitrogen, Life Technologies) and purified with RNeasy microkit columns (Qiagen, Netherlands) as previously published^{25 (link)}. The electropherograms from the bioanalyzer and RIN values showed satisfying RNA quality, as previously published^{25 (link)}. Samples chosen for RNAseq were of satisfying quality with a small percentage of contamination by maternal decidual and nucleated blood cells (~ 2%). Furthermore, the most abundantly expressed genes identified in the term placenta were ones known to be involved in placental function, with good overlap with transcripts identified in previous studies.

Total RNA was isolated from whole abdominal aortas with TriReagent (Sigma-Aldrich, St. Louis, MO) following the manufacturer’s instructions from all groups of rats. An additional round of purification was performed with RNeasy Microkit columns (Qiagen, Venlo, the Netherlands). RNA quality was assessed using RNA 6000 nanochips on the Agilent 2100 bioanalyzer (Agilent Technologies, Amsterdam, the Netherlands), and all samples showed intact 18 S/28 S bands.

C57BL/6 mice harboring a reduced altered Schaedler Flora (ASF⁴; n = 6) or ASF⁴ mice colonized with M. schaedleri MCS for 10 days (n = 6) were sacrificed. The cecum was washed in phosphate-buffered saline (PBS) to remove contents and stored in RNAlater (Qiagen). RNA was purified from cecal tissue samples using TRIzol (Life Technologies, Inc., Carlsbad, CA, USA) followed by RNeasy Microkit columns (Qiagen, Venlo, the Netherlands). RNA quality was assessed on the Agilent 2100 bioanalyzer (Agilent Technologies, Amsterdam, the Netherlands). RNA was labeled using an Affymetrix WT plus reagent kit and hybridized to GeneChip Mouse Gene 1.1 ST arrays (Affymetrix, Santa Clara, CA). Sample labeling, hybridization to chips, and image scanning were performed according to the manufacturer’s instructions. Microarray analysis was performed using the MADMAX pipeline (92 (link)). Quality control was performed, and all arrays met our criteria. A custom annotation that combines all individual probes for a gene (93 (link)) was used. Expression values were calculated and normalized using the robust multichip average (RMA) method (94 (link)), and significant differences were assessed using the paired intensity-based moderated T statistic (IBMT) (95 (link)). Pathway analysis was performed by gene set enrichment analysis (96 (link)); upstream regulator analysis (Ingenuity) was also performed.

Total RNA was extracted using RNeasy Micro kit columns (QIAGEN) and DNase-treated according to the manufacturer’s instructions (Ambion AM1907). RNA (500 ng) was reverse-transcribed using random primers and SuperScript II Reverse Transcriptase (Thermo Fisher Scientific). Control reactions were always performed in the absence of reverse transcriptase and used for RT-qPCR in parallel to cDNA to verify that there was no preexisting DNA contamination. cDNA was diluted in nuclease-free water, and gene expression levels were quantified using RT-qPCR using the QuantStudio 5 Real-Time PCR System (Applied Biosystems) or the LightCycler 480 System (Roche). SYBR Green Fast PCR Mastermix (Life Technologies) was used. Cycle threshold (CT) values for the test genes were normalized against those of Gapdh or Cox6a1 using the –ΔΔCt method to calculate fold change. See the Supplementary Materials for primer sequences.