title: RNA-Seq of Ribosomal prophiling in mice 2h post Hind-Paw Formalin injection
design: RNA-Seq of Ribosomal prophiling in mice 2h post Hind-Paw Formalin injection
library_name: S3 pkus trt_s
library_construction_protocol: Sample preparation: For sequencing experiments, 5 mice were pooled for each sample. Three samples of each conditions were prepared. Immunoprecipitation was performed based on the protocol developed by Knight and colleagues (Knight et al., 2012). The mice were sacrificed 2 hours after formalin injection and quickly dissected in ice cold freshly prepared buffer B (1xHBSS(Life Technologies, 14185-045), 4mM NaHCO3(AppliChem, 131638.1211), 2.5mM pH7.4 HEPES(Roth, 9105.4), 35mM Glucose(Merck, 08337), 100 µg/ml cycloheximide(Sigma, C7698)). The tissue was snap froze and kept in -80ºC. The tissues were kept frozen until homogenization. Homogenization: Tissue was resuspended in 1350µL freshly prepared buffer C (10mM pH7.4 HEPES, 150 mM KCl(Sigma, P9541), 5 mM MgCl2(Sigma, M2670), 1x PhosSTOP phosphatase inhibitor tablet, 1x cOmplete™ Protease Inhibitor Cocktail, 2mM DTT(Sigma, 10197777), 100U/ml RNasin(Promega N2515), 100 µg/ml cycloheximide) and homogenized at 4ºC. The homogenates were centrifuged at 2000g for 10 minutes at 4ºC and the supernatants (~1mL) were transferred to new tubes on ice. 90µL 10% NP40 and 90µL 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC, Avanti Polar Lipids, 850306, 100mg/0.69mL, freshly prepared) were added to each tube. The new mix was centrifuged at 17000g for 10 minutes at 4ºC and the supernatant was collected in a clean tube. A small aliquot of 90 the supernatant was taken for western blot (“Ly”) and the rest were used for immunoprecipitation. Antibody coating: 3µg of pS6 Cell Signaling 5364 antibody and 50µL Protein A Dynabeads (Invitrogen 1001D) were used for each immunoprecipitation. Antibody was incubated with Dynabeads in 1mL buffer A (0.1% TritonX/PBS) for 10 minutes at room temperature with gentle rotation. The coated beads were equilibrated in buffer C till immunoprecipitation. Immunoprecipitation: Immunoprecipitation was performed by applying the supernatant to pS6 coated protein A Dynabeads and rotated at 4ºC for 10 minutes. After immunoprecipitation, the liquid were collected for western blot (“SN”). The beads were washed for four times in 500µL buffer D (10mM pH7.4 HEPES, 350mM KCl, 5mM MgCl2, 2mM DTT, 1% NP40, 100 U/ml RNasin, and 100 µg/ml cycloheximide). During the third wash, the beads were transferred to a new tube and let to incubate at room temperature for 10 minutes. Phospho-ribosomes were eluted with 350µL RLT (or 50µL 2x laemmli for western blot, “IP”). The eluates were immediately processed to RNA extraction for test qPCR or deep sequencing. RNA were extracted from IP eluates. RNA extraction was performed using RNAeasy micro kit (QIAGEN, 74004), following the manufacturer's instruction. The elution volume was 10µL. The RNA was reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher, 18080044) and Oligod(T)23VN (NEB, S1327) following the manufacturer's instruction for SuperScript First-Strand Synthesis System (Thermo Fisher). Library preparation, barcoding and sequencing: Library preparation and barcoding were performed following the standard protocol of EMBL Genecore (Nextera XT DNA Library Preparation Kit, FC-131-1096 and Nextera XT Index Kit v2 Set A, FC-131-2001), using 125pg amplified cDNA as input.