GSM1525107: 13156_CM4G; Mus musculus; RNA-Seq
Total RNA was isolated from cells using the RNeasy Micro Kit (Qiagen).  Briefly,  FACS isolated cells were collected in 200ul of RLT buffer (provided by manufacturer).   The cells were quickly frozen on dry-ice and allowed to thaw on ice.  After the cells were thawed, 50ul of SDS-lysis buffer (50mMTric-HCL pH 7.4/0.05%SDS) was added, the cells were vortexed and an equal volume of 70% ETOH was added.  The cells were voxtexed,  placed on a Qiagen column and centrifuged for 1min at 10,000rpm.  The column was washed by adding 350ul of RW1 and centrifuged for 1min at 10,000rpm.  The extracted RNA was then subject to a 15min on-column DNase digest following the manufacture's protocol.  After DNase digestion, the column was rinsed with a series of washes including: 350ul of RW1, 500ul RPE and 500ul of 80% ETOH.  After the washes, the column was centrifuged at 10,000 rpm for 5min to ensure the removal of residual ETOH.  The RNA was eluted by adding 16ul of RNAse-free H20 and incubated at room temperature for 2min.  The Qiagen column was placed into a 1.5ml eppendorf tube and centrifuged at 10,000 rpm for 1min to collect the RNA. 2ng total RNA was amplified using the Ovation RNA-Seq System v2 (NuGEN #7102).  The libraries were prepared with the Nextera DNA Sample Preparation kit (Illumina Technologies).  50 ng of cDNA was suspended in Tagment DNA Buffer, and tagmentation (fragmentation and tagging with the adaptors) was performed with the Nextera enzyme (Tagment DNA Enzyme), incubating at 55C for 5 min.  Tagged and fragmented cDNA was purified with DNA Clean & Concentrator-5 (Zymo Research).   The libraries were prepared by PCR with Nextera PCR Master Mix, PCR Primer Cocktail and 2 Nextera Indexes (N7XX, and N5XX) according to the following program: one cycle 98C for 30 s (one cycle), 5 cycles of 98C for 10s, 63C for 30s, 72C for 3min.  The purified DNA was captured on an Illumina flow cell for cluster generation.  Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.