Libraries prepared from human blood RNA with standard QuantSeq Library Prep Kit and protocol display distinct peaks in bioanalyzer traces at 197 bp, 212 bp, 222 bp, 235 bp, and 312 bp, which correspond to abundant globin mRNA library fragments (HBA1, HBA2, and HBB). These peaks are reduced in libraries prepared with the RS-Globin Block, Homo sapiens module (RS-GBHs), as shown below in Figure 1 (50 ng whole blood total RNA).
Globin Block libraries can also display: a longer library size distribution, and/or, peaks present at larger molecular weights (~400 bp and higher).
Figure 1: Bioanalyzer traces for human QuantSeq FWD libraries prepared with (+Globin Block) and without (Standard) the RS-Globin Block solution (RS-GBHs, Cat. No. 070.96). Replicate libraries were prepared from 50 ng of whole blood (WB) RNA with the Standard QuantSeq FWD protocol (blue and red traces), versus QuantSeq +Globin Block (green and turquoise traces). RNA was isolated using the SPLIT RNA Extraction Kit without red blood cell lysis (Lexogen). Grey arrows indicate major globin peaks reduced in +Globin Block Libraries.
Pig blood libraries prepared with RS-Globin Block, Sus scrofa (RS-GBSs) also show an altered peak profile compared to libraries prepared with standard QuantSeq RS (see Figure 2 below).
Figure 2 | Bioanalyzer traces for pig QuantSeq FWD libraries prepared with (+Globin Block) and without (Standard) the RS-Globin Block solution (RS-GBSs, Cat. No. 071.96). Replicate libraries were prepared from 100 ng of whole blood (WB) RNA with the Standard QuantSeq FWD protocol (blue and red traces), versus QuantSeq +Globin Block (green and turquoise traces). RNA was isolated using the Preserved Blood RNA Purification Kit + Dnase I Kit (Norgen Biotek). Grey arrows indicate major globin peaks reduced in +Globin Block Libraries.
Globin reduction is best evaluated prior to sequencing by preparing control libraries from the same RNA sample using the standard QuantSeq protocol, alongside libraries prepared with QuantSeq and RS-Globin Block.