Illumina HiSeq 2500 paired end sequencing; Group 3 innate lymphoid cells amplify YAP1-driven small intestinal repair
Group 3 innate lymphoid cells amplify YAP1-driven small intestinal repair
B6.129P2(Cg)-Rorctm2Litt/J (Ror gamma t-GFP) mice were crossed to Lgr5-GFP-IRES-creERT2 (Lgr5-GFP) mice to generate a Ror gamma t-deficient Lgr5 reporter strain. Lgr5GFP/WT x Ror gamma tGFP/GFP (Ror gamma t-deficient) and Lgr5GFP/WT x Ror gamma t+/+ (Ror gamma t-sufficient) mice received two i.p. injections with Methotrexate (MTX) to induce small intestinal damage. Dosages were: day -1: 120 mg/kg, day 0 60 mg/kg. Mice were terminated at days 1 and 4 after the last MTX injection. Mice used: B6.129P2-Lgr5tm1(cre/ERT2)Cle/J; B6.129P2(Cg)-Rorctm2Litt/J or B6.129P2-Lgr5tm1(cre/ERT2)Cle/J. Isolated small intestines were opened longitudinally and washed with cold PBS. 5 mm pieces were washed with cold PBS, incubated in EDTA (2 mM) at 4°C for 30 min and resuspended in PBS. Crypt-enriched sediments were passed through a 70 μm cell strainer and centrifuged at 200 g for 2 min to separate the crypts from single cells. Crypts were incubated with 1 ml of TrypLE Express (Gibco) + Dnase I (Merk Millipore) at 37°C for 10-15 min until crypt dissociation was observed. Single cell suspensions were filtered through 40 μm cell strainer and labeled with conjugated antibodies.  EpCAM1+Lgr5-GFPhi crypt cells were sorted to purity on a FACS AriaIII.  cDNA was prepared using SMARTer Ultra Low RNA kit (Clontech Laboratories) for Illumina Sequencing following the manufacturer's protocol. The Agilent 2100 Bio-analyzer and the High Sensitivity DNA kit were applied to determine the quantity and quality of the cDNA production.
Experimental Factor: compound
methotrexate; first dose 120 milligram per kilogram; second dose 60 milligram per kilogram
Experimental Factor: phenotype
wild type phenotype
Experimental Factor: time
1
Experimental Factor: stimulus
none