PDAP1 RIP in UV-crosslinked human T cells

Human CD4+ Total memory cells (CD4+CD25-CD45RA-CCR7+/-) were activated for 5d with anti-CD3/anti-CD28 antibodies. About 40*10^6 cells were then irradiated twice with 254nm UV light at 0.2J and then lysed in RIPA buffer. 1mg of cell extract was incubated on a rotating wheel ~16 h at 4°C with anti-PDAP1 antibody. PDAP1:mRNA complexes were immunoprecipitated with protein-G dynabeads 4hrs at 4°C and then treated with proteinase K. RNA was then purified using TRI-reagent and RNA zymospin columns. Three independent donors were used for this experiment. Inputs were also sequenced. Total RNA was sequenced in paired-end mode. This experiment is assessing direct targets bound by PDAP1 in human T cells. Overall design: Immunoprecipitation of PDAP1 protein with 1mg of human CD4 T cell extract and sequencing of the PDAP1-associated RNA.