GSM5766803: QKI7 input rep2 [oe.2.in]; Homo sapiens; OTHER
GSM5766803
The ribosomes from the cell lysates were collected by ultracentrifugation and the ribosome footprints (17-34nts) were recovered from polyacrylamide TBE-Urea gel. The end structures of the RNA fragments were repaired by T4 polynucleotide kinase (T4 PNK, M0201S, New England Biolabs): (1) 3' end healing reaction (10 μl): 7 μl RNA sample, 1 μl 10×T4 PNK buffer, 1 μl T4 PNK, and 1 μl SUPERase In RNase Inhibitor were mixed and incubated for 1 h at 37 ℃; (2) 5'-end phosphorylation: 1 μl 10 mM ATP and 1 μl extra T4 PNK were added to the reaction and the mixture was kept for 30 min at 37 ℃. The RNA was purified by ethanol precipitation. The libraries were constructed after depletion of rRNA, and sequenced on Illumina Hiseq2500 with single end 51-bp read length.