5C of p0wt_kidney
Mus musculus
Single cells were crosslinked with 1% formaldehyde for 10 min at room temperature. The crosslinked DNA was lysed and digested with HindIII. The DNAs were then ligated with T4 DNA ligase and purified with phenol-chloroform. 500 ng of the ligated DNA was mixed with 1 μg of salmon testis DNA (Sigma). 1.7 fmol of each 5C primer and 1 μl of 10×5C annealing buffer (NEBuffer 4) were added into the samples to make a total volume of 10 μl. Denaturing was performed by incubating samples at 95 °C for 5 min followed by annealing at 48 °C for 16 h. The DNA was then ligated by Taq DNA ligase (NEB) in the Taq DNA Ligase Reaction Buffer (NEB) at 48 °C for 1 h followed by incubation at 65 °C for 10 min to stop the reaction. 6 μl of ligated products was amplified by PCR using KOD-Plus-Neo (TOYOBO) with specific primers in 25 ul of volume and purified with MinElute PCR Purification Kit (QIAGEN).
strain
C57BL/6
isolate
The kidney of p0 mouse was dispersed by 0.0625% collagenase (Sigma) treatment in DMEM supplemented with 10% FBS for 20 min at 37 C with rotating at 700 rpm. Cells were then filtered through a 100 um cell strainer (BD) to obtain single-cell suspension.
age
p0
sex
not determined
tissue
kidney
biomaterial_provider
Qiang Wu, Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, No.800, Dong Chuan Rd. Shanghai, 200240
collected_by
Yuanxiao Tang
collection_date
2015-10-18
genotype
wild type
geo_loc_name
China:Shanghai
isolation_source
not collected
BioSampleModel
Invertebrate