Insoluble material was pelleted at 12000g at 4C for 10 min. 1mL of supernatant was added to 9mL of dilution buffer. 300uL of this was set aside as an “input” sample. 2ug of TRA-1 antibody UMN163 was added to the remaining sample and incubated in a 15mL conical with rotation at 4C o/n. Samples were spun at 15000g at 4C for 15 min and supernatant was decanted into a fresh 15mL conical tube. 50ul of blocked 50% PAS beads were added and incubated with rotation at 4C for 1 hr. PAS beads were pelleted at 1000g for 30 sec and supernatant was removed. Beads were then washed once in each of the following buffers; low salt buffer, high salt buffer, LiCl buffer, Morohashi RIPA, DOC lysis buffer, and TE. Beads were then transferred to a fresh tube and washed again with TE. Each wash consisted of adding 1mL of 4C buffer, inverting for 30 seconds, spinning at 1000g at 4C for 30 sec, and then discarding the supernatant. After the final wash, residual TE was removed with a small bore pipette tip. 130uL of elution buffer was added to each sample and incubated with rotation at RT for 15 min. Beads were spun down at 1000g for 30 sec and 100uL of supernatant was transferred into a new 1.5mL tube. An additional 100uL of elution buffer was added to each sample and incubated with rotation at RT for an additional 15 min. Again, beads were spun down at 1000g for 30 sec and another 100uL of supernatant was transferred into the same 1.5mL tube, making 200uL total volume per sample. At this point, 200uL of the “input” sample was processed in parallel to the IP samples for the rest of the protocol. 10uL of 4M NaCl was added to each sample and incubated o/n at 65C. Samples were then cooled to RT, and 1uL of 10 mg/mL RNAse A was added to each sample and incubated at 37C for 30 min. Then 4uL of .5M EDTA, 8uL Tris (pH=6.5), and 1uL of PCR grade proteinase K were added to each sample and incubated at 50C for 1 hr. Samples were then purified using Qiagen PCR purification columns and eluted in 50-100uL of Qiagen EB. DNA were blunt-ended, A-overhanged, and then ligated with adaptors. DNA libraries were amplified by PCR with single-end primers. Amplicons in the size range between 200 and 500 bp were recovered from agarose gel.