Dataset Information

Microproteomics of PCBP2 biomolecular condensates

ABSTRACT: PCBP2 condensates were isolated by FAPS. To identify the PCBP2 condensates signature, we performed a mass spectrometry analysis and compared the pre-sorted and post-sorted fractions.For each sorting experiment, cells were cultured to approximately 80-90% confluence in three 15-cm dishes. Cells were pelleted in PBS by centrifugation at 1,000 × g for 3 min at RT and immediately flash-frozen in liquid nitrogen overnight. Unless otherwise stated, the following steps were conducted at 4°C or on ice. Cell pellets were resuspended in lysis buffer (50 mM Tris [pH 7.4], 1 mM EDTA, 150 mM NaCl, 0.2% Triton X-100), containing 65 U/mL RNase inhibitor and EDTA-free protease inhibitor cocktail. To facilitate lysis, the extracts were passed 20 times through a 25G syringe needle, with a total incubation time of 20 min. Lysates were spun at 200 x g for 5 min to remove nuclei. To digest remaining DNA contaminants, the supernatants were treated with RQ1 RNase-free DNase for 30 min at RT, and then centrifuged at 10,000 x g for 7 min. The resulting pellets were resuspended in 2 mL of lysis buffer, and the organelle-enriched fraction obtained at 10,000 x g was designated as the pre-sorted fraction. From this fraction, PCBP2-biomolecular condensates were sorted on a cell sorter (Bigfoot, Invitrogen) equipped with a 100 μm nozzle and operating at a pressure of 30 psi. Particles were identified based on their forward-scattered light (FSC) and mCherry fluorescence, utilizing a 561 nm excitation laser and a 615/24 nm bandpass filter. The sorting window for PCBP2-biomolecular condensates was specifically set to exclude mCherry-SH-SY5Y fluorescent particles while retaining mCherry-labeled PCBP2 condensates. The mCherry-PCBP2 fraction represented 8-10% of total events and was subsequently collected. These fractions were centrifuged at 10,000 × g for 7 min, and the pellets were then analyzed as the post-sorted fractions.

ORGANISM(S): Homo Sapiens

SUBMITTER: Guojun Chen

PROVIDER: PXD055459 | iProX | Fri Aug 30 00:00:00 BST 2024

REPOSITORIES: iProX

ACCESS DATA

Similar Datasets

Project description:This series represents a group of five samples (from sigmoid colon biopsies of clinically normal Patients A-E) profiled by a publicly available cDNA-based platform (GPL284) and a commercial oligonucleotide-based platform (GPL91). Sample RNA extraction: Biopsies taken during endoscopic analysis were immediately snap-frozen in liquid nitrogen and at no time prior to or during subsequent sample manipulation were the samples allowed to thaw. Standard laboratory procedures to eliminate the presence of RNases, including the use of RNase free plastic-ware, heat baked glassware, molecular biology grade chemicals and DEPC-treated solutions, were adopted. Frozen biopsies were crushed to a fine powder under liquid nitrogen using a manual crusher with a teflon head (Omnilab) and total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturers protocol, with minor modifications that improved RNA yields and purity in our hands. In brief, crushed biopsy samples were lysed in an appropriate volume of Buffer RLT (typically 600 µL/20 mg tissue). To facilitate complete lysis, samples were allowed to stand at room temperature for 5-10 min before vortexing. The lysate was homogenized by addition to a QIA shredder column (Qiagen) and centrifuged at 14,000 rpm for 2 min. The resulting supernatant was centrifuged at 14,000 rpm for 3 min to pellet any cell debris. One volume of 70% ethanol was added to the cleared lysate, added to the RNeasy column and centrifuged at 10,000 rpm for 15 sec. Bound RNA was washed with the addition of RW1 buffer to the column and allowed to stand at room temperature for 10 min followed by centrifugation at 10,000 rpm for 15 sec. DNase 1 incubation mix (Stock: 273 kunits; RNase-free DNase Set, Qiagen) was added directly to the spin column membrane and digestion of any contaminating genomic DNA was carried out at room temperature for 30 min. The column was washed with RWI buffer and the DNase treatment step was repeated. The bound RNA was washed with RW1 buffer and centrifuged at 10,000 rpm for 15 sec three times, followed by two additional wash and centrifugation steps with RPE buffer. Finally, purified RNA was eluted off the column with RNase-free water. RNA concentration and purity was determined spectrophotometrically at A260/A280 and was determined to be intact as assessed by formaldehyde gel electrophoresis. The presence of genomic DNA contamination was assessed by PCR amplification of GAPDH using standard PCR conditions and the following primers: GAPDH_F2-5’- ACCCACTCCTCCACCTTTGAC-3’, GAPDH_R2- 5’-CTGTTGCTGTAGCCAAATTCGT-3’. RNA was re-treated with DNase if necessary. Upon confirming the quality of the RNA (intact and free of genomic DNA), 15 µg of total RNA from each of the five biological replicates was used for expression screening on either the Affymetrix or the clone-based filter platform. Keywords: parallel sample

Dataset Information

Microproteomics of PCBP2 biomolecular condensates

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets