GSM9630470: HBoV1_HAE_Day14; Homo sapiens; RNA-Seq
Total RNA was extracted from the HBoV-infected and uninfected HAE cells using RNeasy Mini Kit (Qiagen) with DNase I on-column treatment. RNA sequencing method was designed based on the Drop-seq protocol described in (Macosko EZ et al. Cell 2015). Briefly, 10 ng of RNA was mixed with Indexing Oligonucleotides. After 5 minutes of incubation the RNA was combined with RT mix (1 x Maxima RT buffer, 1 mM dNTPs, 10 U/µl Maxima H- RTase, 1 U/µl RNase inhibitor and 2.5 µM Template Switch Oligo). Samples were incubated for 30 minutes at 22°C and 90 minutes at 42°C. The constructed cDNA was amplified by PCR. The PCR products were pooled together in sets of 12 samples containing different Indexing Oligos and purified with Agencourt AMPure XP Beads according to the manufacturer's instructions. The 3'-end cDNA fragments for sequencing were prepared using the Nextera XT (Illumina) tagmentation reaction. The reaction was performed according to manufacturer's instruction, with the exception of the P5 SMART primer that was used instead of S5xx Nextera primer. Each set of 12 samples that was pooled after the PCR reaction was tagmented with a different Nextera N7xx index. Subsequently, the samples were PCR amplified and purified twice using Agencourt AMPure Beads. The libraries were sequenced on a Illumina NextSeq 500, with a custom primer producing read 1 of 20 bp and read 2 (paired end) of 50 bp. Sequencing was performed at the Functional Genomics Unit of the University of Helsinki, Finland.