7500 fast real time pcr system

Genomic DNA was extracted. SYBR Green-based quantitative real-time PCR analysis (Roche Applied Science) were performed with the 7500 fast Real-Time PCR System (Thermo Fisher Scientific). The forward primer was designed to end with the predicted cleavage site and thus it will not amplify mutant DNA with unmatched 3’. The primer sequences for analysis of Pik3cg gRNA1-mediated genome editing and Vegfr2 gRNA3-mediated genome editing were listed in Table S2.
To determine if QPCR can distinguish DNA with single bp deletion or insertion from wild-type DNA, mutant CRISPR^CDH5 plasmid DNA (CRISPR^Del or CRISPR^Ins plasmids) were made by replacing the wild-type Cas9 fragment with a mutant Cas9 fragment containing single base deletion or insertion using BglII and EcoRV sites. A forward primer matching the wild-type sequence was used to amplify wild-type fragment versus mutant fragment with 1 bp deletion. A forward primer with the 3′insertion nucleotide was used to amplify the mutant fragment with 1bp insertion versus wild-type fragment.

TRIzol^™ Solution (Thermo Fisher Scientific^®) was used for extracting Total RNA from cells and tissues according to the manufacturer’s instructions. Total RNA was quantified by NanoDrop^™ ND-1000 (Thermo Fisher Scientific^®) or Quibit^™ 4 (Thermo Fisher Scientific^®). 50 ng or RNA was reverse transcribed using the cDNA synthesis and gDNA removal QuantiTect^® Reverse Transcription Kit (Thermo Fisher Scientific^®). Real time PCR was performed using the iTaq^™ Universal SYBR^® Green Supermix (Bio-Rad Laboratories, Inc.), 10 ng of cDNA and on a QuantStudio^™ 5 Real-Time PCR System or a 7500 fast real time PCR system (both from Thermo Fisher Scientific^®). Relative gene expression was calculated using the comparative Ct method, normalized to GAPDH. Primers are listed in Supplementary Table 3. Primers were obtained from Integrated DNA Technologies, Inc.

RNA was extracted from sorted microglia using an RNeasy micro kit (Qiagen) according to the manufacturer’s instructions. cDNA was prepared from total RNA by reverse transcription with ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). qRT-PCR was performed with a 7500 Fast Real-Time PCR System (Thermo Fisher Scientific) using Fast SYBR Green Master Mix (Thermo Fisher Scientific). The following primers were used: Apoe forward: 5′-GGCAAAGCAACCAACCCTG-3′; Apoe reverse: 5′-CAGTGCCGTCAGTTCTTGTG-3′; Axl forward: 5′-CAAGAGCGATGTGTGGTCCT-3′; Axl reverse: 5′-TCTCACTGTTCTCCACCCCT-3′; Clec7a forward: 5′-GGGATCAGAGAAAGGAAGCCA-3′; Clec7a reverse: 5′-AGGAAGGCAAGGCTGAGAAAA-3′; Itgax forward: 5′-GCGTGGAGAACTTTGATGCT-3′; Itgax reverse: 5′-CTTGGTGTCTCTGTGCCCTC-3′; Ccl5 forward: 5′-CAGTCGTGTTTGTCACTCGAA-3′; Ccl5 reverse: 5′-AGAGCAAGCAATGACAGGGA-3′; Cxcl10 forward: 5′-CCACGTGTTGAGATCATTGCC-3′; Cxcl10 reverse: 5′-TCACTCCAGTTAAGGAGCCC-3′; Tnf forward: 5′-GATCGGTCCCCAAAGGGATG-3′; Tnf reverse: 5′-ACTTGGTGGTTTGCTACGAC-3′; Il6 forward: 5′-CACTTCACAAGTCGGAGGCT-3′; Il6 reverse: 5′-CTGCAAGTGCATCATCGTTGT-3′; Gapdh forward: 5′-GCAAGGACACTGAGCAAGAGA-3′; and Gapdh reverse: 5′-AGGCCCCTCCTGTTATTATG-3′. Results were normalized to Gapdh. Fold-changes in gene expression were calculated using the 2^−ΔΔCt method.