APEX-seq of time course of P-body and Stress Granule transcriptomes during stress
APEX-seq of time course of P-body and Stress Granule transcriptomes during stress
APEX cell lines were treated with DMEM + 10% FBS containing 0.5mM NaAsO2 (Sigma Aldrich, #S7400-100G) for 0, 15 or 40 mins. Recovery timepoints were treated for 40mins with 0.5mM NaAsO2, then washed twice with PBS and placed in DMEM + 10% FBS for another 60 mins or 120 mins. Following protocol as described previously9,10, samples were treated with DMEM containing 500mM biotin-tyramide (Iris Biotech, #LS-3500.0250) ± 0.5mM NaAsO2, 30 mins before ending the stress incubation. Cells were incubated at 37˚C, 5% CO2 between treatments. 1mM H2O2 (Sigma) was then added to cells for exactly 1 min (except for unlabelled controls). Cells were washed three times with quenching solution (5 mM Trolox (Sigma), 10 mM sodium ascorbate (Sigma), 10 mM sodium azide (Sigma) in PBS), then prepared for RNA extraction or fixed for imaging. Two P10 plates per cell line per timepoint underwent APEX proximity-labelling during stress (see 4.3.2). After quenching washes, each plate was treated with 3mL quenching solution + 4uL RNase inhibitors (RiboLock, ThermoFisher #EO0382), to preserve RNA. Cells were then harvested by scraping and centrifuged at 300xg for 5 minutes at 4˚C to remove quenching buffer. Cells were lysed using RNeasy plus mini kit (Qiagen, 74134) following manufacturer instruction with minor modifications according to Fazal et al. 50. After elution, total RNA concentration and purity were measured with NanoDrop and integrity with TapeStation using electronic ladder.  40µg of total RNA was diluted into 125µl water to pulldown RNA biotinylated by the APEX domain. 10µL DynabeadsTM MyOneTM Streptavidin C1 magnetic beads (#65001) per 25 µg RNA were used. Beads were washed three times with Binding & Washing buffer (5mM Tris-HCl, Ph = 7.5; 0.5 mM EDTA; 1M NaCl; 0.1% Tween 20), twice in 500μL A buffer (100 mM NaOH; 50mM NaCl) and once in 500μL B buffer (100mM NaCl). Bead were resuspended in B buffer with 3uL Riboblock RNAse Inhibitor. RNA mix was added to beads and incubated on rotator for 2hr at 4˚C. After incubation, beads were washed 3 x with B&W buffer. Digestion buffer (330µL 10X PBS, pH = 7.4; 330µL 20% N-laurylsarcosine sodium solution (Sigma #L7414); 66µL 0.5M EDTA; 16.5µL 1M DTT; 357.5 water) with Proteinase K (Ambion) was applied for 1 hour at 42˚C then another hour at 55˚C to remove biotinylated RNA from beads. Supernatant was collected and treated with RNA Clean & Concentrator-5 kit (Zymo Research, R1013), with an additional centrifugation step. Elution was performed with 2 x 6µl RNase-free water. Samples were placed in PCR tubes on dry ice for transport to sequencing facility.  RNA pulled down during biotinylation was sent to Australian Genome Research Facility (AGRF, https://www.agrf.org.au/). Stranded Total RNA with Ribo-Zero Plus Library prep was performed by the facility with 15 cycles of PCR.