Rna nanoprep kit

FACS sorted cells were directly suspended in Trizol, and total RNA was extracted (Rio et al., 2010 (link)). RIN values for all EKLF+/+ and EKLF-/- samples were between 9.1 and 9.8. Poly-A library preparations of biological triplicate samples were analyzed by 100 nt single reads on an Illumina HiSeq 2500 or Illumina Novaseq, 60–90 million reads per sample. For F4/80+ EKLF/GFP+ population, the low cell numbers led us to use an Agilent RNA Nanoprep kit (#400753) for isolating reasonably good-quality RNA (RIN ~7). RNA-Seq data has been submitted to the Gene Expression Omnibus.

Quantitative RT-PCR was performed to analysis the gene expression status by using gene-specific primers (Table 1). Specifically, RNA was extracted from retinal cells using RNA Nanoprep kit (Agilent Technologies, Santa Clara, CA), and reverse transcribed with the Reverse Transcription System (Promega, Madison, WI) to synthesize cDNA. Quantitative PCR was performed in the Rotor-Gene Q Cycler (Qiagen, Valencia, CA) using the SYBR GREEN PCR Master Mix (Qiagen, Valencia, CA). For each gene, relative expression was calculated by comparison with a standard curve, following normalization to the housekeeping gene β-actin expression chosen as a control.Table 1

List of RT-PCR primers

Gene	Forward	Reverse
β-Actin	5′-CGT AAA GAC CTC TAT GCC AA-3′	5′-AGC CAT GCC AAA TGT GTC AT-3′
TNF-α	5′-CAA AAT TCG AGT GAC AAG CCT G-3′	5′-GAG ATC CAT GCC GTT GGC-3′
VEGF	5′-AAC CAT GAA CTT TCT GCT CTC TTG-3′	5′-GCC TGG CTC ACC GCCTTG GCT TGT C-3′
ICAM-1	5′-CCC TGT CAG TCC GGA AAT AA-3′	5′-GAT GAC TTT TGA GGG GGA CA-3′
PLA2	5′-CAG CAA GGA UCC UCG CUA U-3′	5′-CAG AAU GCU UCC AAU CGU A-3′

cDC subsets were sorted from the MLN of WT mice in steady state or following i.p. injection of αCD40 (25 μg; Biolegend) and LPS (20 μg; Sigma-Aldrich) or Poly(I:C) (100 μg; InvivoGen) using a FACSAria III Cell Sorter (BD Bioscience). Briefly, MLN CD11c⁺ cells were enriched using anti-CD11c magnetic cell separation beads (Miltenyi Biotec) and then stained for surface markers prior to sorting. Total RNA was isolated with an RNA Nano-prep kit (Agilent), cDNA was prepared and amplified using Ovation Pico WTA systemV2 (TECAN), and quantitative PCR to quantify p28 expression was performed with Kapa SYBR FAST (Sigma-Aldrich). The following primers were used: b-actin sense, 5′-CCG​GGA​CCT​GAC​AGA​CTA-3′; b-actin antisense, 5′-GTT​TCA​TGG​ATG​CCA​CAG​GAT-3′; p28 sense, 5′-ATC​TCG​ATT​GCC​AGG​AGT​GA-3′; and p28 antisense, 5′-GTG​GTA​GCG​AGG​AAG​CAG​AGT-3′ (Hooper et al., 2017 (link)).