noninfected_m464_b1
Protocols: RNA was extracted (see nucleic acid extraction protocol) from small intestinal epithelium (see below) from mice with citrobacter infection (see treatment protocol) and not, a library was created (see nucleic acid library construction protocol) and sequenced paired end 150 bp (see nucleic acid sequencing protocol)Crypts from distal colon were isolated according to a slight modification of a published protocol (Sato 2013). In summary, the distal colon of mouse was washed in ice-cold PBS and opened longitudinally. A microscopy cover slip was used to gently scrape of excess mucus before the colon was cut into 2-4 mm pieces. The fragments were resuspended in 30 mL ice-cold PBS and pipetted up and down with a 10 mL pipette. Supernatant was discarded and this step was repeated 5-10 times until the supernatant was clear. The fragments were incubated in 10 mM EDTA in PBS at 4 C for 30 min with gentle rocking. The supernatant was removed, 20 mL of ice-cold PBS was added and the fragments were pipetted up and down. This washing step was repeated until the crypt fraction appeared as seen in a microscope. The 1-3 consecutive crypt fractions were passed through a 70 um cell strainer and collected into a FCS coated 50 mL tube. The crypts were spun down at 300 xg for 5 min and washed once in 10 mL ice cold PBS at 200 xg for 5 min. References:Sato, Toshiro and Hans Clevers (2013). “Primary Mouse Small Intestinal Epithelial Cell Cultures”. In:Epithelial Cell Culture Protocols: Second Edition. Ed. by Scott H. Randell and M. Leslie Fulcher. Methods in Molecular Biology. Totowa, NJ: Humana Press, pp. 319-328.isbn: 978-1-62703-125-7.doi:10.1007/978-1-62703-125-7_19.527url:https://doi.org/10.1007/978-1-62703-125-7_19. The mice studies for Citrobacter rodentium infection were approved by Norwegian Food Safety Authority (FOTS ID: 11842). Briefly, C. rodentium was grown at 37 C in Luria- Bertani (LB) medium supplemented with chloramphenicol (30 ug/ml) overnight. 6-8 weeks old C57BL/6 mice were orally gavaged with a culture of 10e8 - 10e9 CFU per mouse delivered in a volume of 100 uL of sterile PBS. Mice remained infected with C. rodentium for six days. To determine the infection level, fecal samples were collected on day 5, homogenized in sterile PBS with a FastPrep homogenizer. After serial dilution of fecal samples, homogenates were plated on chloramphenicol resistant agar plates and the plates were counted after 24 h of growth at 37 C. After 6 days of infection, mice were euthanized by CO2 inhalation, parts of distal colon tissue w RNA was extracted with Quick-RNA Microprep Kit according to the manufacturers instructions (Zymo, R1050). Library preparation was done with NEB Next(R) Ultra(TM) RNA Library Prep Kit
ENA-FIRST-PUBLIC
2021-05-01T00:23:35Z
ENA-LAST-UPDATE
2020-06-03T20:36:11Z
External Id
SAMEA6872472
INSDC center name
Dept. of Clinical and Molecular Medicine Norwegian University of Science and Technology (NTNU)
INSDC first public
2021-05-01T00:23:35Z
INSDC last update
2020-06-03T20:36:11Z
INSDC status
public
Submitter Id
E-MTAB-9184:noninfected_m464_b1
age
72
broker name
ArrayExpress
common name
house mouse
developmental stage
adult
genotype
wild type genotype
individual
J
organism part
crypt of Lieberkuhn of large intestine
replicate
1
sample name
E-MTAB-9184:noninfected_m464_b1
sampling site
distal colon
scientific_name
Mus musculus
sex
female
strain
C57BL/6