GSM2628253: GB0892_GFPN-rep1; Mus musculus; OTHER
Total RNAs of sorted cells were extracted using QIAzol (Qiagen) and purified with miRNeasy micro kit (Qiagen) and DNase set (Qiagen). Purified RNAs were reverse transcribed using PrimeScript Reverse Transcriptase (Takara) with an oligo dT primer. The cDNAs were amplified by VNC-specific and T7 primers and fragmented to 200 to 500bp by sonication. The DNA ends were repaired using End-It DNA End-repair Kit (Epicentre), followed by treatment with Klenow Fragment exo- (New England Biolabs) to generate a protruding 3’A base used for adaptor ligation. Following ligation of a pair of top-Phops-oligo-17bp/T7MmeI-18bp adaptors, the fragments between 250-650bp were isolated from agarose gel. Then the DNA was amplified by MmeI-SD1/2/3 and T7 primers to add an MmeI recognition site at both ends, followed by digestion with MmeI (New England Biolabs). After treatment with Klenow Enzyme (New England Biolabs), indexed libraries were prepared with a Multiplexing Sample Preparation Oligonucleotide Kit (Illumina) according to the user's manual. The fragments between 250-500bp were idolated for sequencing on an Illumina next generation sequencing platform.
GEO
GSM2628253