DDX6_B2_a10_APEX
Homo sapiens
Protocols: APEX labelling in living cells The APEX protocol was carried out as described previously. Briefly, after the release of the second treatment with Thymidine (cc) and at the indicated time points to capture each cell cycle stage, APEX labelling was performed by changing to fresh media containing 500uM biotin-phenol (Iris Biotech), followed by incubation of the cells at 37C and 5% CO2 for 30 minutes. H2O2 (Sigma Aldrich) was then added to each well to a final concentration of 1 mM and incubated for exactly 1 minute. The reaction was then quenched by replacing the medium with an equal volume of 1x Quench buffer (5mM Trolox(Sigma-Aldrich), 10mM sodium ascorbate (Sigma-Aldrich), and 10mM sodium azide (Sigma-Aldrich) in PBS). Cells were washed two more times with 1x quench buffer before fixation (imaging), or PBS followed by cell collection (RNA-seq).  Streptavidin pull-down of APEX-labelled RNA After the second wash with 2X quench buffer in the APEX labelling protocol, cells were harvested by scraping them in 2X quench buffer including 4μL Ribolock RNase inhibitor (Thermo Fisher). The cells were centrifuged for 5 minutes at 200xg at 4 °C. After aspirating the quench buffer, cells were lysed and processed using the RNeasy plus mini kit (Qiagen) following the manufacturer's protocol with minor modifications, including addition of ß-mercaptoethanol to the RLT lysis buffer, washing with the RWT Buffer (Qiagen) instead of RW1 buffer, and application of the on-column DNaseI digestion (Qiagen). The RNA was eluted in RNase-free water and quantified using Nanodrop. Equal amounts of RNA were aliquoted before the streptavidin pull-down of RNA.  To enrich biotinylated RNAs, 10 μl each of Dynabeads™ MyOne™ Streptavidin C1 magnetic beads (Thermo Fisher) were used per 25 μg of RNA. The beads were washed 3 times in 500μL B&W buffer (5 mM Tris-HCl, pH = 7.5, 0.5 mM EDTA, 1 M NaCl, 0.1% TWEEN 20 (Sigma Aldrich)), followed by 2 times in 500μL Solution A (0.1 M NaOH and 0.05 M NaCl), and 1 time in 500μL Solution B (0.1 M NaCl). Beads were then incubated with purified RNA (50μg) in 0.05M NaCl with 4μl/400μl Ribolock RNase Inhibitor (Thermo) at a final volume of 800μl at 4°C for 2 hours with rotations. Following the incubation step, beads were washed 3 times with B&W buffer, followed by the elution of RNA from the beads by incubation with Proteinase K (Ambion) on the beads. Beads were incubated 1 hour at 42C with Proteinase K buffer (final concentrations), and 1 extra hour at 55C for enzyme inactivation. Beads were placed in the magnetic rack and the RNA-containing supernatant was collected. RNA was further cleaned and concentrated using the RNA clean and concentrator 5-kit (Zymo Research) following manufacturer's instructions. RNA concentration was determined by Nanodrop.  HeLa cells from 24-well plates were collected in PBS 1x with Ribolock RNase inhibitor (ThermoFisher) and centrifuged for 5 minutes at 200xg at 4 °C. After aspirating the PBS, cells were lysed and processed using the RNeasy plus mini kit (Qiagen) following the manufacturer's protocol. The RNA was eluted in RNase-free water and quantified using Nanodrop. For RT-qPCR experiments, first-strand cDNA was generated by using the SuperScript VILO Master Mix with ezDNase Enzyme (ThermoFisher) according to the manufacturer's protocol. qPCR was performed in a volume of 10 uL, using 4uL of diluted DNA and 6 ul of master mix, including the Fast Advance Master mix (ThermoFisher) and the two Taqman probes (Gene of interest and GAPDH), following the recommendations using the QuantStudio 5 (ThermoFisher). Gene expression was calculated using the 2(-∆∆CT) method using GAPDH as the housekeeping gene, and the non-treated cells (WT) as reference.  RNA pulled down during biotinylation was sent to Australian Genome Research Facility (AGRF, https://www.agrf.org.au/) for sequencing. Stranded Total RNA with Ribo-Zero Plus Library prep was with 15 cycles of PCR.
ENA first public
2026-01-01
INSDC center name
Garvan Institute BABS UNSW
INSDC status
public
Submitter Id
E-MTAB-14037:DDX6_B2_a10_APEX
broker name
ArrayExpress
cell_line
HeLa
cell_type
epithelial cell
collection date
not collected
common name
human
developmental stage
adult
disease
cervical adenocarcinoma
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
uterine cervix
replicate
rep2
sample group
DDX6_unsynch
sample name
E-MTAB-14037:DDX6_B2_a10_APEX
scientific_name
Homo sapiens