ChIP-qPCR analysis of ARNTL binding

Enteric ILC3 from adult C57BL/6J mice were isolated by flow cytometry. Cells were fixed, cross-linked, lysed and chromosomal DNA-protein complex sonicated to generate DNA fragments ranging from 200-400 base pairs as previously described ^{2 (link)}. DNA-protein complexes were immunoprecipitated, using LowCell# ChIP kit (Diagenode), with 1μg of antibody against ARNTL (Abcam) and IgG isotype control (Abcam). Immunoprecipitates were uncross-linked and analysed by quantitative PCR using primer pairs flanking ARNTL putative sites (E-boxes) in the Ccr9 locus (determined by computational analysis using TFBS tools and Jaspar 2018). Results were normalized to input intensity and control IgG. Primer sequences were: A: F-CATTTCATAGCTTAGGCTGGCATGG; R-CTAGCTAACTGGTCTCAAAGTCCTC; B: F-GCCTCCCTTGTACTACCTGAAGC; R-TCCCAACACCAGGCCGAGTA; C: F-AGGGTCAATTTCTTAGGGCGACA; R-GCCAAGTGTTCGGTCCCAC; D: F-TCTGGCTTCTCACCATGACCACT; R-TCTAAGGCGTCACCACTGTTCTC, E: F-TTTGGGGAATCATCTTACAGCAGAG; R-ATTCATCCTGGCCCTTTCCTTCTTA; F: F-GCTCCACCTCATAGTTGTCTGG; R-CCATGAGCACGTGGAGAGAAAG; G: F-GGTCGAATACCGCGTGGGTT; R-CCCGGTAGAGGCTGCAAGAAA; H: F-AGGCAAATCTGGGCCTATCC; R-GGCCCAGTACAGAGGGGTCT; I: F-GGCTCAGGCTAGCAGGTCTC; R-TGTTTGGCCAGCATCCTCCA; J: F-ACTCAGAGGTGCTGTGACTCC; R-AGCTTTAGGACCACAATGGGCA.

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Godinho-Silva C., Domingues R.G., Rendas M., Raposo B., Ribeiro H., Alves da Silva J., Vieira A., Costa R.M., Morais-Barbosa N.L., Carvalho T, & Veiga-Fernandes H. (2019). Light-entrained and brain-tuned circadian circuits regulate ILC3 and gut homeostasis. Nature, 574(7777), 254-258.

Publication 2019

LowCell# ChIP kit IgG isotype control

Adult Antibody Arntl C57bl mice Ccr9 Cells Chip Chromosomal Flow cytometry Isotype Primer Protein

Corresponding Organization :

Other organizations : University of Lisbon

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Variable analysis

independent variables

Antibody used for immunoprecipitation (ARNTL and IgG isotype control)

dependent variables

Enrichment of DNA fragments containing ARNTL putative binding sites (E-boxes) in the Ccr9 locus, measured by quantitative PCR

control variables

Input DNA intensity
IgG isotype control immunoprecipitation

controls

Positive control: ARNTL antibody immunoprecipitation
Negative control: IgG isotype control immunoprecipitation

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