480 2 light cycler

Two paired-end libraries were prepared from 1.1 μg of gDNA using Illumina TruSeq DNA Preparation Kit, following Illumina’s standard protocol (Paired-end Library Preparation Kit, Illumina, SanDiego, CA, USA). Shearing of gDNA was done using Covaris S series (Covaris, MS, USA). Following end repair, A-tailing, and adaptor ligation, DNA in the 500–600 bp range was purified from a 2% agarose gel. DNA was then PCR enriched for a total of ten cycles. Proper DNA size was then confirmed with the Agilent Bioanalyzer, followed by qPCR quantification with Roche Light Cycler 480 II and Kapa Biosystems reagents.
Cluster generation was performed on an Illumina cBot and the libraries were sequenced on an Illumina HiSeq 2000 following the Paired-End protocol. Sequences can be accessed at NCBI SRA, with accession number SRA092047. The rest of our analysis was initiated from the FASTQ files provided by Illumina's downstream analysis CASAVA software suite.

For total RNA extraction, equal volume of ethanol was added to a sample in TRIzol, and then loaded onto a filter column provided by the miRNeasy Mini Kit (Qiagen, Valencia, CA, USA). The RNA extraction protocol was then performed according to the manufacturers’ instruction, with the exception of two-time DNase treatments (once on-column, and once prior to cDNA synthesis) to ensure minimal DNA contamination. One microgram (1 μg) of extracted RNAs was reverse transcribed according to instructions provided by the Invitrogen™ SuperScript® III First-Strand Synthesis System (Thermo Fisher Scientific, Waltham, MA, USA). The temperature settings for reverse transcription were 5 min at 25 °C, 60 min at 50 °C, and 15 min at 70 °C, and the samples were then chilled on ice. For qPCR, KAPA SYBR® FAST qPCR Master Mix (Kapa Biosystems, Wilmington, MA, USA) was used, followed by real-time PCR in Roche Light Cycler® 480II for 10 min 95 °C preheat, then 40 PCR cycles of 10 s at 95 °C, 10 s at 50 °C, and 10 s at 70 °C. The primers used for qPCR are listed in Additional file 1: Table S6.

RNA expression levels for housekeeping genes (Hmbs, PGK1), Iba1 and targeted floxed genes (Tgfb1^fl/flor Alk5^fl/fl) were determined by Reverse-Transcribed quantitative real-time PCR. RNA was extracted from FACS sorted YFP+ or tdTomato+ cells using a RNAqueous-Micro Total RNA isolation kit (AM1931, ThermoFisher Scientific). Total RNA was treated with RNase free DNase and cDNA was then generated using iScript cDNA synthesis kit (1708890, BioRad). cDNA levels for Hmbs (hydroxymethylbilane synthase), pGK1 (phosphoglycerate kinase 1) and various target genes were determined, using specific primer/probe sets by quantitative RT-PCR using a Roche Light Cycler II 480 (Roche, Basel, Switzerland). Relative expression level was calculated using the delta Ct method compared to Hmbs as a reference gene and expressed as fold change compared to the average of WT cells for each individual gene. Primers and carboxyfluorescein (FAM) labeled probes used in the quantitative RT-PCR for each gene are listed in the oligonucleotide section. To selectively detect the presence of the floxed region in the TGFb1^fl/fl or the ALK5^fl/fl microglia, the probe based qRT-PCR reactions were selected so that the forward and reverse primers span the exon junctions of the floxed exon and a neighboring exon therefore ensuring the loss of amplification in the case of a successful cre-lox mediated recombination.

Dopaminergic cultures were harvested at DIV28 for total RNA extraction following instructions from the manufacturer (RNAqueous, Ambion). Total RNA (1 μg) was treated with RQ‐1 Rnase‐free Dnase I and reverse transcribed into cDNA using the Superscript III reverse transcriptase kit. cDNA levels for GAPDH, HPRT1, Nurr1, TH, DAT, VMAT2, Drd2, ptx3, mu, kappa, and delta opioid receptors were determined by specific universal probe library primer probe sets (Roche) using Roche Light Cycler II 480. Primers and FAM‐labeled probes used in the quantitative RT‐PCR for each gene are listed in Table 2. Relative expression level was calculated using ΔΔCt method compared to GAPDH as a reference gene. All of the results were obtained with triplicate samples for each line/conditions/experiment and were combined from at least two independent experiments.

Stool samples were collected throughout the 14-day antibiotic treatment period, and DNA was isolated using the DNeasy PowerSoil DNA isolation kit (Qiagen) according to the manufacturer’s instructions. Stool samples were collected throughout the 14-day antibiotic treatment period, and fecal DNA was isolated using the DNeasy PowerSoil DNA isolation kit (Qiagen) according to the manufacturer’s instructions. qPCR for bacterial 16S (UniF340 [5′-ACTCCTACGGGAGGCAGCAGT-3′] and UniR514 [5′-ATTACCGCGGCTGCTGGC-3′]) was performed on a Roche 480II LightCycler and used to determine the microbial load in the intestines of these mice. The relative bacterial burden was calculated using the ΔΔC_T method, and values were expressed as log fold changes from the values for untreated mice.