title: GSM1399475: Mmus Suz12-KO RNA Differentiation 24h rep. 2; Mus musculus; RNA-Seq
library_construction_protocol: mESCs were grown in 10 cm plates as described above, washed once with 1X PBS, lysed with 4 mL of TRIzol Reagent (Ambion) and split in 4 x 1 mL. Each aliquot was mixed with 200 µL of Chloroform:Isoamyl alcohol (24:1) (CI) and centrifuged at full speed for 15 min at 4°C. Supernatants were extracted twice with 500 µL of Phenol:CI (125:24:1) (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, resuspended with 10 mM Tris-HCl, pH 8.0. Aliquots from the same samples were pooled. RNA purity and amount was checked using a NanoDrop spectrophotometer. Expected yields range from 100 to 200 µg. 30 µg were further purified using the RNeasy MinElute Cleanup Kit (Qiagen) according to the manufacturer's instructions to remove small RNA (i.e. < 200 nt). RNA integrity was checked using the RNA 6000 Nano Kit with a 2100 Bioanalyzer (Agilent).    Depletion of 18S and 28S rRNA was performed on 5 µg of each sample using the RiboMinus Human/Mouse Transcriptome Isolation Kit (Ambion) according to the manufacturer's instructions. RNA fragmentation was performed using the NEBNext Magnesium RNA Fragmentation Module (New England Biolabs) as follows. 600 ng was mixed with 50 µL of 1X RNA Fragmentation Buffer, incubate at 94C for 20 min, transferred on ice and mixed with 5 µL of 10X RNA Fragmentation Stop Solution. Depletion in 18S and 28S rRNA (before fragmentation) and fragmentation efficiency was checked using the RNA 6000 Pico 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 RNA Clean & Concentrator-5 Kit (Zymo Research) using the manufacturer protocol for small fragments and eluted in 14.5 µL.    RNA end-repair was performed by mixing samples with 0.5 µL of Murine RNase Inhibitor, 2 µL of 10X T4 Polynucleotide Kinase Buffer (New England Biolabs), 1 µL of 10U/µL T4 Polynucleotide Kinase Enzyme, incubating at 37C for 30 min, mixing with 2 µL of 10 mM ATP and incubating at 37°C for 30 minutes. Samples were purified using the RNA Clean & Concentrator-5 Kit as before and eluted in 8 µL. RNA amount was quantified using a NanoDrop spectrophotometer (40 to 50 ng/µL). 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: GSM1399475