GSM5395561: Ribotag3_WT_M476; Mus musculus; RNA-Seq
The pull-down of Rpl22HA/HA was performed as described before (Sanz, E., Yang, L., Su, T., Morris, D.R., McKnight, G.S., and Amieux, P.S. (2009). Cell-type-specific isolation of ribosome-associated mRNA from complex tissues. Proc Natl Acad Sci U S A 106, 13939-13944.) using the Pcp2Cre:Rpl22HA/HA and Pcp2Cre:Tie2flox/flox:Rpl22HA/HA mouse lines. In short, cerebellar tissue samples were homogenized on ice in 10% w/v polysome buffer. Samples were centrifuged for 10min at 12.000rpm at 4°C, supernatant was collected and 10µl were kept as input. Remaining sample was incubated o/n at 4°C with anti-HA antibody-coated magnetic beats. Magnetic beads were washed with ice-cold HS-buffer and finally re-suspended in RLT buffer from RNeasy® Mini Kit (Qiagen; 74104). Library preparation and sequencing were performed by BGI Genomics, China.