GSM8766533: Inner hair cells, P0, WT, ATAC-Seq, rep1; Mus musculus; ATAC-seq
Atoh1-GFP positive inner hair cells (IHCs) and Atoh1-GFP; Insm1-GFP-Cre and R26-TdTomato positive sensory outer hair cells (OHCs) were FACS-sorted from P0 cochleae into 500 uL 1x PBS with 2% FBS. Cells were centrifuged at 700g for 20 min at 4°C. The supernatant was removed and cells were lysed in 20 uL lysis buffer (10mM Tris HCl, 5mM MgCl2, 10% DMF, 0.2% NP-40) at 4°C. 30 uL reaction buffer (10mM Tris-HCl pH 8, 5 mM MgCl2, 10% DMF, 3000 u Tn5 transposase from Segil lab) was added, and sample was incubated at 37°C for 30 min. Fragments were purified using the MinElute Reaction Cleanup Kit (Qiagen), and DNA fragments were eluted into 23 uL elution buffer from kit. A 5-cycle PCR was peformed using a mix with 20 uL DNA fragments solution, 2.5 ul primer 1 (25 uM), 2.5 uL primer 2 with barcode (25 uM), 0.3ul SYBR Green and 25 uL 2x NEB Next High-Fidelity PCR Master MIix to give a final volume of 50 uL, using an intial elongation step of 3 min at 72°C followed by 5 cycles of 98°C for 10s, 63°C for 30s and 72°C for 1 min, and a final elongation step at 72°C for 3 min. 5 ul of each sample was taken and run in a QuantStudi 7 Flex Real-Time PCR System (Applied Biosystems) with an intitial denaturaiton at 98°C for 1 min, followed by 40 cycles at 98°C for 10s, 63°C for 30s and 72°C for 1 min.  The number of cycles to reach 1/4 saturation point was determined for each sample, and the remaining 45 uL of the sample was run with an initial denaturation temperature at 98°C for 1 min, followed by the required number of cycles to reach 1/4 saturation point at 98°C for 10s, 63°C for 30s and 72°C for 1 min. 45 uL AMPure XP Reagent (Beckman Coulter) was added then added to PCR products following the reaction. Tubes were placed on a magnetic beads separation rack, and the supernatant was discarded. Beads were washed twice using 200 uL 80% ethanol, and DNA was eluted from the beads using 22 uL nuclease-free water. Samples were sent to the NUSeq core facility at Northwestern University for size selection for fragments between 200bp and 1kb, and sequencing using the Illumina NextSeq 500 sequencer. Samples were sequenced using 37bp paired-end reads to a depth of about 50 million reads.