| Construction Protocol | Crosslinked cell pellets were resuspended in 500 µl lysis buffer with 1x cOmplete Protease Inhibitor tablet (Roche, Basel, Switzerland), and then 700 µl of 500 µm acid-washed glass beads were added. The cells were vortexed for 12 minutes total, in cycles of 2 minutes shaking and 2 minutes resting on ice. The lysate was pelleted and resuspended in fresh lysis buffer, and then sonicated for 3x10 min runs on a Diagenode Bioruptor (Diagenode, Liège, Belgium), on high power with cycles of 30 seconds on and 30 seconds off, to an average of ~300 bp. The sonicate was cleared by centrifugation, and the resulting supernatant was split into an input aliquot (1/20 of IP volume) and two halves for the IP and mock-IP (BSA instead of antibody). For each sample, 5 ug of anti-TAP (Thermo Fisher Scientific, Waltham, MA; #CAB1001, Lot #RL240352) was used with 10 ul Dynabeads Protein A (Thermo). The antibody or BSA was incubated with pre-washed beads for 2 hours and then washed twice in fresh lysis buffer before being added to the lysate and then incubated overnight. After washing the beads four times in lysis buffer and eluting, the eluate was reverse crosslinked and then treated with RNase A and Proteinase K. DNA was purified using Zymo ChIP DNA Clean & Concentrator (Zymo Research, Irvine, CA) and eluted in 30 µl water. ChIP-seq libraries were prepared using the Accel-NGS 2S Plus DNA Library Kit (Swift Biosciences, Ann Arbor, MI), from equal volume pools of the biological replicates, either 1 ng total from input samples or 21 ul of total IP sample. Input and IP libraries were amplified for 6 and 7 cycles, respectively. |
FTP
