title: Mettl3_PAR-CLIP_rep2
design: RAW264.7 cells treated with VSV for 12h were cultured in medium supplemented with 200 M 4-thiouridine for 14h, and then irradiated once with 400 mJ/cm2 at 365 nm using the UVP for crosslinking. Cells were harvested in lysis buffer and rotated for 30 min at 4 C. Cell debris was removed by centrifugation and the supernatant was digested by 1 U/l RNase T1 at 22 C in a water bath for 8 min and cooled on ice for 5 min. Then the lysates were incubated with antibody overnight at 4 C. Washed magnetic Protein A beads were added to the mixture and incubated for another 2 hr, and the beads were then washed three times with IP wash buffer. Beads were digested with 10 U/l RNase T1 again at 22 C in a water bath for 8 min, cooled on ice for 5 min, then washed three times in high salt wash buffer, resuspended in 100 l dephosphorylation buffer, and incubated with 0.5 U/l calf intestinal alkaline phosphatase (CIP, NEB) for 10 min at 37 C with gentle rotation. Beads were then washed twice with phosphatase wash buffer  with 3 min rotation. Beads were resuspended with 200 l proteinase K buffer , and incubated at 55 C for 2 hr. RNA was extracted with Phenol: Chloroform. Sequencing libraries were constructed using the SMARTer smRNA-seq kit (Clontech). Sequencing was performed on an Illumina HiSeq X Ten instrument with paired end 150-bp read length.