title: E-MTAB-6560:GF_ZT13_Input_7
attributes.tag: Alias
attributes.value: E-MTAB-6560:GF_ZT13_Input_7
attributes.tag: Broker name
attributes.value: ArrayExpress
attributes.tag: Description
attributes.value: Protocols: Mice C57BL/6J mice were purchased from Charles River. Germ-free mice generated from C57BL/6J mice were obtained either from CNRS TAAM UPS44 Orléans, France, or from the Gnotobiology Platform of the Institut Pasteur and kept in isolators. Conventional mice were kept in specific pathogen- free conditions and all the mice used were female. Mice were housed in 10h (dark)/ 14h (light) cycles. All animal experiments were approved by the committee on animal experimentation of the Institut Pasteur and by the French Ministry of Agriculture. For generating ex-GF mice, 10-12 fecal pellets were collected from 4 CONV mice housed in the same cage for 2 weeks, resuspended, and added to about 100 mL of drinking water of GF mice that had been water-deprived over night. This procedure was repeated on 3 consecutive days. On Day 4, cecal content of the 4 CONV mice was collected, resupended in 10 mL of PBS, of which 200ul was administered to the pre-colonised mice by oral gavage. Colonisation efficiency was determined in the cecal content of mice after 4 weeks. Antibiotic treatment of mice: For depletion of the gut flora, conventional C57BL/6J mice were treated with antibiotics as described previously22. In brief, after treatment with amphotericin B (0.1 mg/ml; Sigma Aldrich) for 2-3 days, mice were treated with a solution consisting of 10 mg/mL ampicillin, 5 mg/mL vancomycin 10 mg/mL neomycin, 10 mg/mL metronidazol and 0.1 mg/ml amphotericin-B (all Sigma Aldrich) per os every 12h using a gavage volume of approximately 10 mL/kg body weight for 21 days. The depletion was controlled for by quantitative PCR as described. Total RNAs were prepared using the RNeasy maxi kit (Qiagen) and quality-controlled using RNA 6000 Nano assay (Agilent). Ribodepletion was performed using RiboMinus Transcriptome Isolation Kit (Human/Mouse; Thermo Fisher). anti- m6A immunoprecipitation Immunoprecipitation was performed as previously described 38. Briefly, 3 g of rabbit anti m6A antibody (Synaptic Systems) were bound to 25 l washed Protein G Dynabeads (Thermo Fisher) in immunoprecipitation buffer (1x IPP; 150 mM NaCl, 0.1% NP-40, 10 mM Tris-Cl, pH 7.4) for 30 min at room temperature (RT) and washed twice in 1x IPP. Total (liver) or ribodepleted (cecum) RNA was fragmented using the NEBNext Magnesium RNA fragmentation module (New England Biolabs), purified by ethanol precipitation and quality-controlled using the RNA 6000 pico assay (Agilent). Equal amounts of RNA (4 g for ribodepleted and 200 g for total RNA) were denatured for 2 min at 70°C and adjusted to 1x IPP concentration using 2x IPP. RNA was added to the antibody-bound beads and incubated for 2h at 4°C in the presence of murine RNase inhibitor (New England Biolabs). The bound RNA was washed twice with 1x IPP, twice with low salt IPP buffer (50 mM NaCl, 0.1% NP40, 10 mN Tris-Cl pH 7.4), twice with high salt IPP buffer (500 mM NaCl, 0.1% NP40, 10 mN Tris-Cl pH 7.4) and once more in 1x IPP. RNA was eluted using 30 l of buffer RLT (Qiagen). 20 l of MyOne Silane Dynabeads (Thermo Fisher) were washed with RLT and resuspended in 30 l of RLT. Eluted RNA was bound to the beads in the presence of 35 l of absolute ethanol, washed twice in 70% ethanol and eluted in 100 l of H20. RNA was purified and concentrated using RNA clean & concentrator (Zymo research) before proceeding to library preparation. 150 ng of input RNA and the immunoprecipitated RNAs were dephosphorylated using antarctic phosphatase and subjected to T4 PNK treatment (both New England Biolabs).
attributes.tag: ENA checklist
attributes.value: ERC000011
attributes.tag: INSDC center alias
attributes.value: Institut Pasteur - Bioinformatics and Biostatistics Hub - C3BI, USR 3756 IP CNRS - Paris, France
attributes.tag: INSDC center name
attributes.value: Institut Pasteur - Bioinformatics and Biostatistics Hub - C3BI, USR 3756 IP CNRS - Paris, France
attributes.tag: INSDC first public
attributes.value: 2018-04-11T17:01:56Z
attributes.tag: INSDC last update
attributes.value: 2018-03-01T11:58:28Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: SRA accession
attributes.value: ERS2257665
attributes.tag: Sample Name
attributes.value: ERS2257665
attributes.tag: Title
attributes.value: GF_ZT13_Input_7
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: growth condition
attributes.value: germ-free
attributes.tag: individual
attributes.value: GF_ZT13_7
attributes.tag: organism
attributes.value: Mus musculus
attributes.tag: organism part
attributes.value: liver
attributes.tag: replicate
attributes.value: 7
attributes.tag: sequencing
attributes.value: seq4
attributes.tag: sex
attributes.value: female
attributes.tag: strain
attributes.value: C57BL/6J