Ez magna rna immunoprecipitation kit

An EZ Magna RNA immunoprecipitation Kit (Millipore, USA) was applied as directed by the manufacturer. In brief, KYSE510 cells underwent lysis with RIP lysis buffer. The resulting cell lysates were added to magnetic beads linked to anti-Ago2 antibodies (Millipore, USA) or control anti-IgG for overnight incubation at 4 °C. The immunoprecipitated RNA was isolated and assessed by qRT-PCR.

The EZ-Magna RNA immunoprecipitation kit (Millipore, US) was used according to the manufacturer’s specifications. A549 and NCI-H1299 cells were lysed in complete RIP lysis buffer. The cell extract was then incubated with magnetic beads conjugated with Ago2 antibody (abcam, UK) or control IgG (Millipore, US) overnight at 4 °C. The beads were then washed and incubated with 0.1% SDS/0.5 mg/ml proteinase K for 30 min at 55 °C to remove proteins. Finally, the immunoprecipitated RNA was analysed by qRT-PCR analysis.

The RNA pull-down assay was conducted as previously described [23 (link)]. In brief, the biotin-labeled anti-sense DNA probes for LINC01273 were designed and synthesized by Sangon (Shanghai, China). The HCC cell lysates were then collected and incubated with LINC01273 probe at for 2 h at 4°C. RNA complexes were washed with NT2 buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM MgCl₂, and 0.05% NP-40) and incubated with streptavidin-magnetic C1 beads (Cat.65001, Invitrogen) for 0.5 h at 4°C. The enriched RNA was extracted and used for qRT-PCR analysis. And RIP assay was performed using EZ Magna RNA Immunoprecipitation Kit (Cat.17–701, Millipore) according to the manufacturer’s guidelines. The IP antibody was anti-Ago2 (ab186733, Abcam) and anti-YTHDF2 (ab246514, Abcam).

The EZ‐Magna RNA immunoprecipitation kit (Millipore, USA) was used for RIP assay. The lysates of NSCLC cells samples were incubated with magnetic beads conjugated with Ago2 antibody (#ab32381, abcam, UK) or control IgG (Millipore) overnight at 4°C. After washing three times, the beads were incubated with 0.1% SDS/0.5 mg/mL proteinase K for 30 min at 55°C. The immunocomplexes were then analysed by RT‐qPCR.

RIP assays were conducted using the EZ‐magna RNA Immunoprecipitation Kit (Millipore) in accordance with the manufacturer's protocols. In brief, cells were collected and lysed using RIP lysis buffer. The whole‐cell extracts were then incubated with RIP buffer containing magnetic beads conjugated to anti‐AGO2 or control IgG. The beads were washed using washing buffer, followed by addition of proteinase K to digest the proteins. Subsequently, the immunoprecipitated RNAs were isolated and subjected to qRT‐PCR analysis.

The EZMagna RNA immunoprecipitation Kit (17-701; Millipore) was used to investigate whether AP000439.2 could interact potential binding proteins STAT3. 786-O cells were lysed using RIP lysis buffer to harvest cell lysates and 10% lysates were used as input. Then, left cell lysates individually incubated with magnetic beads conjugated with anti-STAT3 (SAB1406670; Millipore) antibody and control IgG (Millipore) at 4 °C for 12 h. After that, the beads were washed three times with RIP buffer. Next, added Proteinase K into tubes and incubated at 55 °C for 30 min to remove the proteins. Finally, the expression of AP000439.2 in purified RNA was detected by RT-qPCR, and U6 was served as control.

The EZ Magna RNA immunoprecipitation Kit (Millipore, USA) was used following the guidelines. Briefly, HCT-116 and HCT-8 cells were lysed in RIP lysis buffer. Magnetic beads were pre-incubated with antibodies for 30 min at room temperature and the cell lysates was immunoprecipitated with beads for 6 h at 4 °C. Then, RNA was purified and detected by qRT-PCR. Antibodies information are listed in Additional file 3: Table S3.