mRNAs in total RNA contain 5
0 phosphate and 3
0 hydroxyl termini
and could easily compete out sRNAs for the pre-adenylated
3
0 adapter, leading to bias toward the recovery of the most abundant
sRNAs in the sRNA library construction. Another issue in the NGS
including sRNA-seq that prevents the precise quantification is that
the ligation of adapters might have bias toward certain RNAs
probably due to inherent RNA secondary structure or chemical
reaction preference as previously reported [12, 13].
Based on the previous methods [7, 11] and our extensive
experience in sRNA studies [14–19], we have optimized our protocol (Fig. 1). Compared to commercial kits and most published
protocols, ours has several unique features: (1) It provides friendly
applied and cost-effective technique to synthesize a large amount of
3
0 pre-adenylated adapters so that sufficient amount of adapters can
Fig. 1 Work flow for the construction of a sRNA library
Identification and Quantification of sRNAs
227
0 phosphate and 3
0 hydroxyl termini
and could easily compete out sRNAs for the pre-adenylated
3
0 adapter, leading to bias toward the recovery of the most abundant
sRNAs in the sRNA library construction. Another issue in the NGS
including sRNA-seq that prevents the precise quantification is that
the ligation of adapters might have bias toward certain RNAs
probably due to inherent RNA secondary structure or chemical
reaction preference as previously reported [12, 13].
Based on the previous methods [7, 11] and our extensive
experience in sRNA studies [14–19], we have optimized our protocol (Fig. 1). Compared to commercial kits and most published
protocols, ours has several unique features: (1) It provides friendly
applied and cost-effective technique to synthesize a large amount of
3
0 pre-adenylated adapters so that sufficient amount of adapters can
Fig. 1 Work flow for the construction of a sRNA library
Identification and Quantification of sRNAs
227