Quantstudio 7 flex

ChIPs for histone modifications and SUZ12 were performed as described previously [57 (link)]. For anti-Flag, BMI1 and RBPJ ChIPs, 4.5x10⁶ cells were incubated for 20 min in 1 ml swelling buffer (25 mM HEPES pH 7.8, 1.5 mM MgCl₂, 10 mM KCl, 0.1% NP-40, 1 mM DTT, 1 mM PMSF, 1 μg/ml aprotinin and 1 μg/ml pepstatin A). Nuclei were resuspended in 1 ml sonication buffer (50 mM HEPES pH 7.8, 140 mM NaCl, 1 mM EDTA, 1% Triton-X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1 μg/ml aprotinin and 1 μg/ml pepstatin A) and sonicated for one hour using a Covaris M220 (75 W peak power, 26 duty cycle, 200 cycles/burst and 6°C set temperature). Thereafter, the ChIP assay kit from Millipore (17–295) was used, according to the manufacturer’s protocol. DNA was cleaned using QIAquick PCR purification Kit (Qiagen) and was assayed by qPCR on QuantStudio 7 Flex (Life technologies). Input DNA was 5% of DNA used in immunoprecipitations and diluted to 2.5% prior to PCR quantification. Enrichment relative to input was calculated using four 5-fold-dilution series and error bars calculated as standard deviations from triplicate PCR reactions for both input and IP. Antibodies used are listed in S3 Table and sequences of the primers used in these assays are listed in S4 Table.

Isolation of total RNA from ear skin and from BMDM cultures was carried out according to standard protocols using TRI reagent (Sigma-Aldrich). cDNA was generated by RevertAid reverse transcriptase (ThermoFisher). Quantitative PCR was performed using SYBR green (Roche) and a QuantStudio 7 Flex (Life Technologies) instrument. The primers used for qPCR were as follows: Il17a, forward 5′-GCTCCAGAAGGCCCTCAGA-3′, reverse 5′-AGCTTTCCCTCCGCATTGA-3′ (67 (link)); Tnf, forward 5′-CATCTTCTCAAAATTCGAGTGACAA-3′, reverse 5′-TGGGAGTAGACAAGGTACAACCC-3′ (68 (link)); Ifnb, forward 5′-AACCTCACCTACAGGGC-3′, reverse 5′-CATTCTGGAGCATCTCTTGG-3′ (42 (link)); Actb, forward 5′-CCCTGAAGTACCCCATTGAAC-3′, reverse 5′-CTTTTCACGGTTGGCCTTAG-3′. All RT-qPCR assays were performed in duplicate, and the relative expression (rel. expr.) of each gene was determined after normalization to Actb transcript levels.