title: GSM1139749: Mettl3 knockdown 4; Mus musculus; OTHER
library_construction_protocol: Total RNA was extracted using Trizol reagent (Invitrogen). RNA was treated with RNAse free DNAse I (Roche) to deplete DNA contamination. PolyA RNA was purified using GenEluteª mRNA Miniprep Kit (Sigma Aldrich) per manufacturer's instruction. PolyA RNA was fragmented using RNA fragmentation kit (Ambion). Two _g fragmented RNA was saved as input. Two hundred _g fragmented RNA was incubated with 3 _g anti-m6A antibody (Synaptic Systems) in RIP buffer (150mM NaCl, 10mM Tris, 0.1% NP40) for 2 hrs at 4 oC, followed by the addition of washed protein A/G magnetic beads (Millipore) and incubation at 4 oC for additional 2 hrs. Beads were washed 6 times in RIP buffer and incubated with 50 _l IP buffer containing 0.5mg/mL to elute RNA. IP'ed RNA was extracted with phenol/chloroform. The Scriptseq v.2 RNA-seq Library Preparation method was performed on 40 ng of RNA for each sample. We followed the protocol given in the Scriptseq v.2 RNA-seq Library Preparation manual (Epicentre, an Illumina company), except DNA Clean & Concentratorª-5 - PCR/DNA clean columns (Zymo Research) were used to isolate the cDNA prior to PCR step. 15 cycles of PCR were performed on the cDNA and the resulting DNA products were purified on 2% E-Gel¨ EX Agarose Gels (Invitrogen). The products were visualized using a blue light transluminator and selected regions of the gel were excised corresponding to 200-400 bp products. The gel slices were dissolved in agarose dissolving buffer and the DNA was isolated on DNA Clean & Concentratorª-5 - PCR/DNA clean columns. The isolated DNA products were then analyzed and quantitated on an Agilent Bioanalyzer 2100.  Samples were pooled and loaded into one lane of a HiSeq2000 v3 flowcell (Illumina) and sequenced for 100 bases with separate 7 base indexing read.
attributes.tag: GEO Accession
attributes.value: GSM1139749