title: GSM5343555: G009_SG_Wills_08_UNI24_S9_R; Xenopus tropicalis; ATAC-seq
library_construction_protocol: At each timepoint, regenerating tadpoles were anesthetized until non-responsive to touch. A scalpel was used to amputate to the regenerated tail tissue. Regenerated tail tips were collected and spun down at 1000xg. Media was taken off and ~60 tails were resuspended in 200 uL of 0.035 mg/ml liberase in PBS (Roche 05401119001). Tails were left to incubate for 20 min at room temperature. A p-200 was then used to pipet the tails up and down gently and break up tail tissue. Once no visible chunks were apparent, cells were spun down for 3 min at 1000xg. Supernatant was disposed and cells were washed with 180 uL of 1xPBS. Cells were spun down for 3 min at 1000xg and supernatant was discarded. Cells were resuspended in 15 uL of TN5 reaction mix using the TN5 enzyme, buffer, and water from the Nextera DNA Sample prep kit (Illumina 15028212). Cells were transposed for 1 hr at 37C. Post transposition, cells were removed from 37C and DNA was purified using the Qiagen MinElute kit (Qiagen 28206). DNA was eluted in 10 uL of buffer EB. Purified DNA was then amplified using the 2xNEB PCR Master Mix (NEB M0541L), a universal forward primer, and an indexed reverse primer from the i7 Illumina series Initial amplification as follows: 72C:5 min, 98C:30 s, then 5 cycles of 98C:10 s, 63C:30 s, 72C:1 min. 5 uL of the initial PCR product was then used for a 15 µl side qPCR reaction to determine how many more cycles to run to stop amplification prior to saturation. The side reaction was carried out as follows: 98C:30 s, then 30 cycles of 98C:10 s, 63C:30 s, 72C:1 min. The number of cycles was determined by taking the range of the starting and ending CT values and dividing that number by 4, then identifying how many cycles the sample had gone through at the derived CT value. The initial PCR reactions were then run for the calculated number of extra cycles and removed from the thermo cycler. Amplified samples were purified with the Qiagen MinElute kit and eluted in 20 uL of buffer EB. 5 uL of purified PCR product were run out on a 5% acrylamide gel in 1xTBE at 100V for ~45 min. Gels were stained with Ethidium Bromide and imaged on the gel imager (BioRad). Samples with sufficient periodic bands, relating to length of DNA to wrap a mono-, di-, tri- nucleosome, were then run out on a bioanalyzer. Library concentrations were taken using the Qubit high sensitivity dsDNA assay. Libraries were pooled by normalizing library concentrations to the lowest concentration and adding equal volumes of libraries for a minimum of 5 ng of each library. Libraries were sequenced on the Illumina Next-Seq platform across one NextSeq 500 High Output Kit v2.5 (150 cycles, Illumina CAT. 20024907) for TSA and respective controls and one Next-seq High output 150 cycle and one Next-seq Mid output 150 cycle kit (150 cycles, Illumina CAT.  20024904) Libraries were sequenced with, paired end, 75 bp reads at the Seattle Genomics facility.
attributes.tag: GEO Accession
attributes.value: GSM5343555