Identifying Circadian Transcripts in Mouse Proximal Tubule

Rationale: To provide a guide for physiological studies on the kidney proximal tubule, we have now created a data resource consisting of expression levels for all measurable mRNA transcripts in microdissected proximal tubule segments from mice as a function of the time of day. This approach employs small-sample RNA-sequencing (RNA-seq) applied to microdissected renal proximal tubules [33769951], including both S1 proximal convoluted tubules and S2 proximal straight tubules. The data are provided as a user-friendly web page at https://esbl.nhlbi.nih.gov/Databases/Circadian-Prox/. Methods: The study was done in mice (male C57BL/6,4-6 weeks) maintained in an environmentally controlled animal room with a 12hr:12hr light:dark cycle. Three animals were selected at each of 6 time points starting at 8 a.m. (lights on, Zeitgeber 0) and then at every-four-hour junctures. Each of thethree animals in at each time point were studied on separate days. They mice were rapidly euthanized, kidneys were perfused with a physiological solution containing collagenase, followed by manual microdissection as described by Wright and Knepper [2074757] to obtain S1 proximal convoluted tubules and S2 proximal straight tubules. The S1 segments were distinguished by their attachments to glomeruli. The S2 segments originated from the outer cortex. After washing by dragging through clean dissection solution, the tubules were processed for small-sample RNA-seq as described by Chen et al. [33769951]. Each time point was represented by 3 samples for both S1 and S2 segments, each from a different mouse. RNA-Seq used the NEBNext(TM) low Input RNA library preparation kit and a NovaSeq6000 DNA sequencer. Transcripts per million (TPM) values were calculated for each sample. The data were filtered to include only transcripts with a maximum TPM>7 to remove noisy, low abundance mRNAs. The data were analyzed using JTK-Cycle (20876817) to detect periodicity. For this analysis, circadian genes were those with a period 20 or 24 hours, and a curve fit significance level of P