title: GSM8127859: MOLM13_shNS_mRNA_rep2; Homo sapiens; RNA-Seq
library_construction_protocol: The ribosomes from the cell lysates were separated in the sugrose gredient by ultracentrifugation and were collected. ribosome protecting RNA fracgments 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 using NEBNext small RNA library prep set for Illumina (NEB) Sequencing was performed using an Illumina®HiSeq 2500 system, utilizing a 51 bp paired-end configuration