GSM1608003: WT ESC CapSeq; Mus musculus; OTHER
Small RNAs of from total RNAs were resolved on a 15% polyacrylamide 7M Urea Gel, along with 2 picomoles of RNA standard (18- and 40-nt) in separate lanes. SYBR gold staining was used to visualize the RNA standards. A gel fragment was excised from the sample lanes in the migration range between the two standards. RNA was eluted from the gel fragment in (0.3M NaCl-TE pH7.5) solution overnight and isopropanol-precipitated with 20mg of glycogen as the carrier. The gel purified RNA was incubated with 0.25 U/µl TAP (Epicentre) and 1U/µl SuperRNaseIN (Ambion) in 10µl reaction containing 1X buffer at 37 oC for 1 hr, followed by phenol-chloroform extraction and isopropanol precipitation. The TAP-treated samples were then incubated with 5µM of 3'-end linker (1, 2, 3 from IDT DNA Technologies), 1 Unit/µl of SuperRNaseIN, 10% DMSO and 1 U/µl T4 RNA ligase (Takara) in 10µl ligation buffer (50mM Tris-Cl pH7.5, 10mM MgCl2,10mg/mL BSA, 10mM DTT). The 3' ligated products were gel purified, eluted, precipitated and then incubated with 5 µM of 5' adapter oligonucleotide with 4nt barcode at the 5' end, 1 Unit/µl SuperRNaseIN (Ambion) and 2 U/µl of T4 RNA ligase in ligation buffer (50mM Tris-HCI pH7.5), 10mM MgCI2, 10mM DTT, 1mM ATP) and 10% DMSO. The ligated products were gel purified and reverse transcribed in a standard 20µl reaction (SuperScript III, Invitrogen). The cDNA was amplified by PCR and purified in a 8% acrylamide gel. PCR products generated for all the samples were sequenced on a Solexa sequencing HiSeq 2000 platform (Illumina, Inc.) and single-end 100 nt reads were obtained. small RNA cloning, 5' and 3' ligation dependent
GEO Accession
GSM1608003