ATACseq of S.rosetta thecates Replicate 2 Lane 2
For transposition, nuclei were pelleted at 1000 x g for 5 minutes at 4C, resuspended in 25 L of 2x TD buffer and 2.5 l of Tn5 transposase from the Nextera DNA Library Prep kit (Illumina, San Diego, CA), and incubated at 37C for 30 minutes. DNA was purified using the Qiagen MinElute kit (Cat. No. 28004) per PCR purification protocol provided by the manufacturer. Transposed DNA was originally amplified and barcoded in a PCR reaction using NEBnext PCR master mix (NEB Cat. No. #M0544) and 1.25 M forward and reverse primers originally described in (Buenrostro et al. 2013), using the following PCR conditions: 72C for 5 minutes; 98C for 30 seconds; and thermocycling at 98C for 10 seconds, 63C for 30 seconds and 72C for 1 minute. To reduce GC and size bias in the PCR, we monitored the PCR reaction using qPCR in order to stop amplification before saturation. To do this, we amplified the full libraries for five cycles, after which we took an aliquot of the PCR reaction and added 10 l of PCR cocktail with Sybr Green at a final concentration of 0.6x. We ran this reaction for 20 cycles to determine the additional number of cycles needed for the remaining 45 L reaction. The libraries were purified using a Qiagen PCR cleanup kit. Libraries were amplified for a total of 10-12 cycles. An additional 0.9X SPRI bead cleanup was performed to eliminate a contaminating 50 bp peak