title: GSM1399488: Mmus E14-WT iTC serum NoDrug 0h 4sU  rep. 1; Mus musculus; RNA-Seq
library_construction_protocol: mESCs were grown in 10 cm plates as described above. Labeling was performed by replacing the old medium with new medium pre-equilibrated at 37°C, 5% CO2 and either freshly supplemented with 150 µM of 4-thiouridine or not (background control). After 10 min of incubation at 37°C, 5% CO2, cells were washed once with 1X PBS, lysed with 4 mL of TRIzol Reagent and split in 4 x 1 mL. Each aliquot was mixed with 200 µL of CI and centrifuged at full speed for 15 min at 4°C. Supernatants were extracted twice with 500 µL of PCI, pH4.5, once with CI and ethanol precipitated with 0.3 M NaCl, 40 μg of RNAse-free glycogen at -80°C for 15 min. After centrifugation at 20,000 x g for 90 min at 4°C, pellets were washed with 70% ethanol, air dried, re-suspended with 10 mM Tris-HCl, pH 8.0. Aliquots from the same samples were pooled. RNA purity and amount was checked using a NanoDrop and RNA concentration was adjusted to 1 µg/µL. 75 µg were further purified using the Micro Bio-Spin P-30 Gel Columns (RNase-free) Kit (Bio-Rad) according to the manufacturer's instructions.     RNA fragmentation was performed using the NEBNext Magnesium RNA Fragmentation Module as follows. 60 µg was mixed with 75 µL of 1X RNA Fragmentation Buffer, incubate at 94C for 20 min, transferred on ice and mixed with 7.5 µL of 10X RNA Fragmentation Stop Solution. RNA integrity (before fragmentation) and fragmentation efficiency were checked using the RNA 6000 Nano Kit with a 2100 Bioanalyzer. After fragmentation, the size of RNA fragments should have a normal distribution peaking at 60 nt. Samples were purified using the Micro Bio-Spin P-30 Gel Columns (RNase-free) Kit as before.    RNA end-repair was performed as follows. Samples were incubated at 65C for 5 min to disrupt RNA secondary structure and transferred on ice, mixed with 90 µL of H2O, 20 µL of 10X T4 Polynucleotide Kinase Buffer (New England Biolabs), 10 µL of 10U/µL T4 Polynucleotide Kinase Enzyme, incubated at 37C for 1 hour, mixed with 5 µL of 100 mM ATP and incubated at 37°C for 1 hour. Each sample was split in 2 x 100 µL aliquots before purification with the RNeasy MinElute Cleanup Kit with the following modified procedure to keep small RNAs. Aliquots were mixed with 350 μL of Buffer RLT (without β-Mercaptoethanol), 550 μL of 100% Ethanol and run through an RNeasy MinElute spin column. Columns were washed once with 500 µL of Buffer RPE (containing Ethanol) and once with 500µL of 80% Ethanol, dried and eluted twice with 14 µL of H2O. Eluates from the same samples were pooled and volumes were adjusted 52.5 µL with H2O. Biotinylation of the 4sU-labeled RNAs was performed by mixing samples with 7.5 µL of 10X Labeling Buffer (100 mM Tris-HCl pH7.5, 10 mM EDTA), 15 µL of 1 mg/mL EZ-Link HPDP-Biotin (Pierce) dissolved in dimethylformamide and incubating at 25°C for 2 hours. Samples were purified with the Micro Bio-Spin P-30 Gel Columns (RNase-free) Kit as before, mixed with 2 µL of 5M NaCl, incubated 65°C for 5 min and placed on ice. After a second purification using the Micro Bio-Spin P-30 Gel Columns (RNase-free) Kit, volumes were adjusted to 200 µL with H2O, samples were incubated at 65°C for 5 min and placed on ice. Biotinylated RNA pull-down was achieved as follows. For each sample, 1 mg of Dynabeads MyOne Streptavidin C1 (Invitrogen) was washed twice with 190 µL of Solution A (0.1 M NaOH, 0.05 M NaCl) for 2 min, twice with 190 µL of Solution B (0.1 M NaCl) and resuspended in 200 µL of 2X SBW buffer (20 mM Tris-HCl pH7.5, 1M NaCl, 0.2 % SDS). Bead suspensions were then mixed with the denatured RNA samples, incubated at 25°C for 1 hour, washed 3 times with 900 µL of 1X SBW buffer, washed 3 times with 900 µL of 10 mM Tris-HCl pH 7.5 and eluted twice with 100 µL of SE Buffer (10 mM Tris-HCl pH7.5, 5 mM EDTA, 0.1 % SDS) at 99°C for 5 min. Eluates were centrifuged at full speed to remove traces of beads, pooled and ethanol precipitated with 0.3 M NaCl, 40 μg of RNAse-free glycogen at -80°C for 15 min. After centrifugation at 20,000 x g for 90 min at 4°C, pellets were washed with 70% ethanol, air-dried and resuspended with 7µL H2O. RNA amount and size was checked using the RNA 6000 Pico Kit. Each sample was supplemented with 10 ng of inactivated carrier RNA consisting of an equimolar mix of both HPLC-purified RNA oligonucleotides 5'-GAUGAAACGAGUACUACCGACC-3' and 5'PHO-GAUGAAACGAGUACUACCGACC-3'ddC (with 5’PHO and 3'ddC stating respectively for 5’ Phosphate and 2',3' dideoxyC modifications). RNA library for Illumina sequencing was prepared using the NEBNext Multiplex Small RNA Library Prep Set for Illumina® (1–12) (New England Biolabs). Multiplex 3’ SR Adaptor ligation was performed as follows. 6 µL containing either 200 ng or 30 ng of RNA (for either RNA-seq or iTC-seq samples respectively) were mixed with 1 µL of 1:5 diluted Multiplex 3’ SR Adaptor, incubated at 70°C for 2 min and transferred to ice. Samples were then supplemented with 10 µL of 2X 3’ Ligation Reaction Buffer and 3 µL of 3’ Ligase Enzyme Mix and incubated at 25°C for 1 hour. RT primer hybridization was performed by mixing samples with 4.5 µL of H2O, 1 µL of 1:5 diluted Multiplex SR RT Primer and by incubating successively at 75C for 5 min, 37C for 30 min and 25C for 15 min. Multiplex 5’ SR Adaptor ligation was done by mixing samples with 1 µL of denatured 1:5 diluted Multiplex 5’ SR Adaptor, 1 µL of 5’ Ligation Reaction Buffer, 1 µL of 5’ Ligase Enzyme Mix and incubating at 25°C for 1 hour. cDNA synthesis was performed by mixing samples with 8 µL of First Strand Synthesis Reaction Buffer, 1 µL of Murine RNase Inhibitor, 1 µL M-MuLV Reverse transcriptase (RNase H-) and incubating successively at 50°C for 1 hour, 70 °C for 15 min and on ice. cDNA samples were purified using the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer's instructions and eluted in 40 µL. cDNA amplification was performed as follows. Samples were mixed with 50 µL of LongAmp Taq® 2X Master Mix, 5 µL of 1:5 diluted Multiplex SR Primer and 5 µL of 1:5 diluted Multiplex Index (X) Primer. PCR was performed in a Thermocycler with the following program: 1 cycle for the initial denaturation step (94°C for 60 s), either 12 or 15 cycles (for either RNA-seq or iTC-seq samples respectively) for the amplification step (94°C for 10 s, 60°C for 30 s, 65°C for 30 s), 1 cycle for the final extension step (65°C for 7 min) and 1 cycle for the cooling step (4°C). PCR product purification was performed using AMPure XP system (Agencourt) as follows. Samples were split in 2 x 50 µL aliquot. Each aliquot was mixed with 90 µL of AMPure XP beads and incubate at room temperature (RT) for 15 min. Beads were washed twice with 200 µL of 80% ethanol for 30 s, air dried and resuspended with 10 µL of H2O. Supernatants from same sample were pooled and DNA amount was quantified using the dsDNA HS Assay Kit with a Qubit Fluorometer (Invitrogen). Expected amount range from either 300 to 450 ng or from 20 to 40 ng (for either RNA-seq or iTC-seq samples respectively). Library purity and integrity was checked using the DNA 1000 Kit with a 2100 Bioanalyzer (Agilent). DNA sizes should have a normal distribution peaking at 180 nt. Equimolar amounts of either 4 RNA-seq samples or 2 iTC-seq samples, with compatible indexes, were pooled for multiplex sequencing. Pool concentration was adjusted between samples and 0.1 to 0.4 pmols were sent to the National High-throughput DNA Sequencing Centre of the University of Copenhagen for 50 bp HiSeq Single Read sequencing using Illumina Sequencing Technology.
identifiers.namespace: GEO
identifiers.id: GSM1399488