title: GSM5105943: TET1 CUT&RUN; Rattus norvegicus; OTHER
library_construction_protocol: Two million cells were harvested by centrifugation (600 g, 3 min) and cells were washed twice in 1 ml Wash Buffer (20 mM HEPES-KOH pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1x Roche cOmpleteTM), followed by addition of 10 µl pre-activated concanavalin A coated magnetic beads (Bangs Laboratories-BP531). Tubes were held on mixing platform at RT for 10 mins, then placed on a magnet stand and pulled off the liquid. Cells were resuspended in 100 µl cold Antibody Buffer and 1 µl antibody (TET1, Active Motif #61943) was added for incubating on nutator overnight at 4ºC. Beads with cells were washed twice in 1 ml Digitonin Buffer (20 mM HEPES-KOH pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1x Roche cOmpleteTM,0.05% digitonin), and resuspended in 50 µl Digitonin Buffer with addition of 2.5 µl CUTANA pAG-MNase (20x stock). After 10 min incubation at RT, samples were washed twice in 1 ml Digitonin Buffer and changed to 50 µl cold Digitonin Buffer. Then tubes were placed in a metal block in ice-water and quickly mixed with 100 mM CaCl2 to a final concentration of 2 mM. The reaction was quenched by the addition of 50 µl Stop Buffer. Cleaved fragments were liberated into the supernatant by incubating the tube for 30 min at 37ºC. DNA fragments were purified by Qiagen MinElute PCR Purification Kit from the supernatant and used for the construction of sequencing libraries. Libraries were prepared for Illumina sequencing with Library Prep for CUT&RUN with NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (E7645). CUT&Tag followed by  Illumina sequencing