Mus musculus
Protocols: All animal experiments were approved by the committee on animal experimentation of the Institut Pasteur and by the French Ministry of Agriculture.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. 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 the drinking water of GF mice. This procedure was repeated on 3 consecutive days. On Day 4, cecal content of the 4 CONV mice was collected, resuspended in 10 mL of PBS, of which 0.2 mL was administered to the pre-colonized mice by oral gavage. Colonization efficiency was determined in the cecal content of mice after 4 weeks. For mono-colonization experiments, germ-free mice were inoculated once with 109 colony-forming units (CFU) of A. muciniphila, L.plantarum or E.coli Nissle and maintained for four weeks. Mono-colonization was monitored by A. muciniphila- (Collado et al., 2007), L.plantarum- (Quere et al., 1997) or E.coli- (Blum-Oehler et al., 2003) specific PCR and controlled by 16S DNA sequencing in cecal content. For depletion of the gut flora, conventional C57BL/6J mice were treated with antibiotics as described previously (Reikvam et al., 2011). In brief, after oral 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 (Reikvam et al., 2011) and the identity of residual bacterial genera determined by 16S DNA sequencing. For enrichment of a small number of genera, mice were treated as above with only 5 mg/mL vancomycin and 0.1 mg/ml amphotericin-B. Tissues used for the analysis were derived from 2-3 independent experiments. 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). mRNA purification was performed using Dynabeads® mRNA Purification Kit for mRNA Purification from Total RNA preps (Thermo Fisher). Absence of ribosomal RNA contaminations was controlled using RNA 6000 Pico assay (Agilent). Immunoprecipitation was performed as previously described (Schwartz et al., 2013). 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), ribodepleted (cecum) or polyA-purified (cecum/liver) 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 (5 g for ribodepleted and polyA-selected 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 mM Tris-Cl pH 7.4), twice with high salt IPP buffer (500 mM NaCl, 0.1% NP40, 10 mM 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). 150 ng of input RNA and the immunoprecipitated RNAs were dephosphorylated using Antarctic phosphatase and subjected to T4 PNK treatment (both New England Biolabs).
ENA-FIRST-PUBLIC
2020-02-12T17:04:42Z
ENA-LAST-UPDATE
2019-06-10T10:43:45Z
External Id
SAMEA5702702
INSDC center name
Institut Pasteur - Bioinformatics and Biostatistics Hub - C3BI, USR 3756 IP CNRS - Paris, France
INSDC first public
2020-02-12T17:04:42Z
INSDC last update
2019-06-10T10:43:45Z
INSDC status
public
Submitter Id
E-MTAB-6560_2:GF_Liver_2019_IP_GF10-T13
broker name
ArrayExpress
common name
house mouse
ip_procedure
IP_2019
library_batch
181215
organism part
liver
phase
Phase_2019
replicate
GF10
sample name
E-MTAB-6560_2:GF_Liver_2019_IP_GF10-T13
scientific_name
Mus musculus
sequencing
seq2019
sex
female
stimulus
GF_T13
strain
C57BL/6J