RNA-seq Library Prep with Globin Depletion

Purified RNA was analyzed on Bioanalyser (Agilent) for quality assessment. RNA samples with RNA Integrity Number (RIN) of more than 6 were selected for the study (RIN ranging from 6.3 to 9.1 and with a median of 7.5) (Supplemental Table 1). cDNA libraries were prepared by Smart-Seq v2 [21 (link)], using a modification of the GlobinLock (GL) method [22 (link)] to block transcription of globin mRNA. Human “DNA 3 long A” and “DNA 3 long B” oligonucleotides (0.6 pmol each) were added to 2 ng of total blood RNA in 2.3 μL, denatured at 95 °C for 30 s, incubated at 60 °C for 10 min for GL oligo hybridization, and held at 42 °C for the loading of the reverse transcriptase (RT) mixture. RT and subsequent steps were according to Smart-Seq v2 with the following modifications: (1) addition of 20 μM template switching oligos (TSO) and (2) use of 200 pg cDNA with 1/5 reaction of Nextera XT Kit (Illumina). The length distribution of the cDNA libraries was monitored using a DNA High Sensitivity Reagent Kit on the LabChip (Perkin Elmer). All samples were subjected to an indexed paired-end sequencing run of 2 × 151 cycles on a HiSeq 4000 system (19 samples/lane; Illumina).