GSM2127109: RNA-Seq mouse 3T3 shLuc(2); Mus musculus; RNA-Seq
[ChIP-seq SELEX] 300ng of the resultant library mouse genomic DNA was run again with the new empty sepharose beads and GST-Osr1 beads, and the process was repeated four times.Eluted DNA was purified by MinElute Reaction Cleanup Kit (Qiagen). The DNA ends were repaired and generated a protruding 3’ A as described above. Following ligation of a pair of Solexa adaptors to the repaired ends, 150 to 400bp fragments were recovered from agarose gel. Then DNA was amplified using the index primers for 18 cycles and the 150-400bp fragments were isolated from agarose gel again. [ChIP-seq SELEX] The libraries enriched at rounds two, three, and four were sequenced using Illumina high throughput sequencing [RNA-seq] For mRNA-seq library prep:  a random primer (invitrogen cat# 48190-011) was used to prime the purified PolyA tailed-RNA and the cDNA synthesis was performed using SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen cat# 11917-010)  according to the users manual. The ds cDNA was subject to sonication in Diagenode's Bioruptor (level M, total of 30 minutes of 20 seconds ON and 20 seconds OFF)  to a size range of 100-400bp. Indexed library was prepared using Illumina's multiplexing sample prep oligonucleotide kit (Ref# 1005709) with Epicentre's End-It DNA End-repair Kit (Epcentre, cat# ER81050) according to the user's manual and Illumina's multiplexing sample preparation guide. [RNA-seq] The library was prepared using Illumina kits as described G. Hu et al. (Nature immunology 2013). The libraries were amplified using a two-step method to preferentially amplify the small DNA fragments derived from the DNA hypersensitive sites and to reduce non-specific amplification of the carrier DNA. The first amplification was done with index primers with the PCR condition: 98°C, 10”; 67°C, 30”; 72°C, 30” for 6 cycles. The second amplification was done with the P5 and P7 primers with the condition: 98°C, 10”; 68°C, 30”; 72°C, 30” for 22 cycles. The fragments between 160bp to 300bp were isolated on E-gel and sequenced on Illumina HiSeq2000. [bPPI-seq] 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). [bPPI-seq] 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 isolated for sequencing on an Illumina next generation sequencing platform. [ChIP-seq] Cells were crosslinked with formaldehyde treatment and chromatin fragmented to 200 to 300 bp by sonication. Chromatin from 2 × 107 cells was used for each ChIP experiment, which yielded approximately 200 ng of DNA. Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. [ChIP-seq] Libraries were prepared according to Illumina's instructions. The ChIP DNA ends were repaired using PNK and Klenow enzyme, followed by treatment with Taq polymerase to generate a protruding 3′ A base used for adaptor ligation. Following ligation of a pair of Solexa adaptors to the repaired ends, the ChIP DNA was amplified using the adaptor primers for 17 cycles and the fragments around 220 bp (mononucleosome + adaptors) isolated from agarose gel. Libraries were sequenced on the Hi-seq following the manufacturer's protocols. As described in Barski et al., 2007 (A. Barski, S. Cuddapah, K. Cui, T.Y. Roh, D.E. Schones, Z. Wang, G. Wei, I. Chepelev and K. Zhao, High-resolution profiling of histone methylations in the human genome, Cell 129 (2007), pp. 823-837).
GEO Accession
GSM2127109