title: GSM7883263: inner medulla, NFAT5-KO, rep3; Mus musculus; ATAC-seq
library_construction_protocol: Fresh kidneys were removed from six mice. A small piece of renal cortex tissue (approx. 2 mm) and the renal inner medulla from each single kidney were extracted. ATAC was performed with 100.000 cells per sample using the ActiveMotif ATAC-seq kit (Carlsbad, CA), according to the manufacturer's instructions (manual version B6) for tagmentation and purification. For the library preparation a total of 10 µl of purified tagmented DNA was indexed and pre-amplified for initial 5 PCR cycles with 1x KAPA HiFi HotStart Readymix and 100 nM unique dual index P5 and P7 primers compatible with Illumina Nextera DNA barcoding, under the following PCR conditions: 72°C for 5 min, 98°C for 30 s, thermocycling for 5 cycles at 98°C for 10 s, 63°C for 30 s and 72°C for 1 min. Subsequently, a qPCR on the LightCycler 480 (Roche) was performed with 1 µl of the pre-amplified material to determine the remaining PCR cycle numbers (8, 9, or 11) to avoid saturation and potential biases in library amplification 33. Purification and double-sided size selection of amplified libraries was done with AMPure XP beads (Beckmann Coulter; starting with a 1.55x volume of XP bead purification, followed by a 0.6x/1.55x double-sided size selection), checked for their quality on a Fragment Analyzer (Agilent) and quantified using qPCR.