J Am Soc Nephrol 2021, PMID: 33769951
Brief Method: kidneys from 6-week-old WT male and female mice (only female proximal tubules were included in this database) were perfused via the left ventricle with perfusion buffer
(135 mM NaCl, 1 mM Na2HPO4, 1.2 mM Na2SO4, 1.2 mM MgSO4, 5 mM KCl, 2 mM CaCl2, 5.5 mM glucose, 5 mM HEPES, pH 7.4).
kidneys were then sliced and dissociated in perfusion buffer supplemented with collagenase B.
Microdissection were performed under the stereomicroscope equipped with on-stage cooling.
After washes in ice-cold PBS, the microdissected renal tubules were transferred to Trizol reagent for RNA extraction. cDNAs were synthesized by SMARTer V4 kit (Clontech) and cDNA libraries were constructed for paired-end sequencing and sequenced on Illumina HiSeq3000 platform. Reads were mapped to mouse Ensembl Genome by STAR and transcript abundances were calculated in the units of transcripts per million (TPM) using RSEM (https://github.com/deweylab/RSEM).
Terminology: PTS1 the initial segment of the proximal convoluted tubule; PTS2 , proximal straight tubule in cortical medullary rays; PTS3 , last segment of the proximal straight tubule in the outer stripe of outer medulla; DTL1 , the short descending limb of the loop of Henle; DTL2 , long descending limb of the loop of Henle in the outer medulla; DTL3 , long descending limb of the loop of Henle in the inner medulla; ATL , thin ascending limb of the loop of Henle; MTAL , medullary thick ascending limb of the loop of Henle; CTAL , cortical thick ascending limb of the loop of Henle; DCT , distal convoluted tubule; CNT , connecting tubule; CCD , cortical collecting duct; OMCD , outer medullary collecting duct; IMCD , inner medullary collecting duct. Microdissected mouse glomerulus transcriptome data can be accessed at Mouse Glomerulus Atlas .
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