GSM9037399: HEK293T cells, ZAK-1-649, untreated, biol rep 2; Homo sapiens; OTHER
Cells at ~70% confluency in a 10cm dish were crosslinked at 254 nM UV, lysed, clarified, and RNAseI digested (Invitrogen AM2295). IP was performed overnight at 4C using 13 uL of anti-FLAG M2 affinity resin (Millipore A2220). Following IP, samples were washed, dephosphorylated with FastAP enzyme (Thermo EF0654) and T4 PNK (NEB M0201L). The 3' RNA adapter was ligated (NEB M0437M). A small amount of sample was run on a Criterion XT 4-12% Bis-Tris gel and transferred to PVDF to perform a diagnostic WB probing for ZAK expression and size. The remaining sample was run on a Criterion XT 4-12% Bis-Tris gel (Bio-Rad 3450124) and transferred to a nitrocellulose membrane and a region was cut corresponding to the ZAK protein size to plus ~75 kDa. Membrane pieces were digested with Proteinase K (NEB P8107S). RNA was purified on a clean and concentrator column and reverse transcribed with SuperScript III (Thermo 18080044). The RNA and free primer was digested and the RT-DNA purified with MyOne Silane beads (Thermo 37002D), and the 5' DNA adapter was ligated (NEB M0437M). Following cleanup, a pilot qPCR was performed. Samples were amplified for the determined number of PCR cycles, gel-extracted for products corresponding to 175-350 bp, and submitted for next generation paired-end sequencing with 2 x 150 bp read length.