Illumina HiSeq 1500 paired end sequencing; Binding of the Zika virus genome to endogenous human transcripts
Binding of the Zika virus genome to endogenous human transcripts
Infected JEG3 cells were washed with Hanks' Balanced Salt solution (Sigma). RNA was crosslinked by irradiating with long wavelength UV in the presence of Psoralen-TEG-azide. This was repeated on a separate population of JEG3 cells without the crosslinking step to form the no-crosslink negative control. JEG3 cells were maintained in Minimum Essential Medium supplemented with 1mM sodium pyruvate, 10% (vol/vol) fetal bovine serum, non-essential amino acids, and 10 U/mL Penicillin and 100 μg/mL Streptomycin. Cells were maintained in 37 degrees Celsius, 5% CO2 conditions and were periodically examined for mycoplasma contaminations. The Zika virus strain PE243 was propagated in Vero cells and kept aliquoted at -80 degrees Celsius. All handling of Zika virus was performed in a contaminant level 2 facility. JEG3 cells were inoculated with Zika virus strain PE243 in complete growth medium for 20 hours. For all samples, 3' DNA adaptors containing 6 nt long unique molecular identifiers (UMIs) were ligated to the RNA products, following a clean-up step with Agencourt RNAClean XP beads (Beckman Coulter Life Sciences). Conversion to cDNA was done with Superscript III (Thermo Fisher Scientific) by using primers complementary to the 3' adaptors. cDNA was purified by Agencourt AMPure XP beads (Beckman Coulter Life Sciences). RNA was extracted from infected cells using the RNeasy RNA isolation kit (Qiagen). Proteins and DNA were cleared by proteinase-K and DNase I treatments respectively. Virus RNA was affinity enriched by complementary biotinylated DNA probes. Extracted RNA was fragmented with RNase III. For the crosslinked samples, the azide group in psoralen was converted to biotin for further enrichment of crosslinked RNA fragments. The enriched RNA was then divided to two aliquots. One aliquot was proximity-ligated and then UV irradiated to reverse the crosslinks, forming the group of libraries labelled as "Psolaren-based crosslinking". The other aliquot was used as a reverse-crosslink control, where UV irradiation was performed to reverse the crosslink prior to proximity ligation. For the no-crosslink controls, proximity ligation was performed prior to the UV irradiation step.
Experimental Factor: protocol
cross-link reversal before ligation