title: CONV_Input_3
organism: Mus musculus
description: 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).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-02-12T17:04:42Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-06-10T10:43:43Z
attributes.tag: External Id
attributes.value: SAMEA5702619
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: 2020-02-12T17:04:42Z
attributes.tag: INSDC last update
attributes.value: 2019-06-10T10:43:43Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6560_2:CONV_Input_3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: ip_procedure
attributes.value: IP_2018
attributes.tag: library_batch
attributes.value: lib1
attributes.tag: organism part
attributes.value: cecum
attributes.tag: phase
attributes.value: Phase_1
attributes.tag: replicate
attributes.value: 3
attributes.tag: sample name
attributes.value: E-MTAB-6560_2:CONV_Input_3
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sequencing
attributes.value: seq7
attributes.tag: sex
attributes.value: female
attributes.tag: stimulus
attributes.value: CONV
attributes.tag: strain
attributes.value: C57BL/6J