GSM1894186: Young whole cells; Homo sapiens; RNA-Seq
RNA was isolated from whole or mitoplast fraction of cultured young and senescent WI-38 cells. To obtain mitoplast fraction, crude mitochondria were first isolated from cultured WI-38 cells.  Briefly, cells were scraped, centrifuged, and washed once with ice-cold PBS.  After subsequent centrifugation, the cell pellets were resuspended in a hypotonic buffer (20 mM HEPES [pH 7.3], 1.5 mM MgCl2, 10 mM KCl), vortexed at low speed for 5 seconds, and incubated on ice for 2 min.  CHAPS buffer (20 mM HEPES [pH 7.3], 8% (w/v) CHAPS) was added and vortexed vigorously 5 times while incubating on ice for 5 min.  Hypertonic buffer (20 mM HEPES [pH 7.3], 420 mM mannitol, 140 mM sucrose, 2 mM EGTA) was then added with equal volume of hypotonic buffer, and gently mixed by inverting the tubes several times.  After centrifugation at 2,000 x g, supernatants were collected and centrifuged again at 2,000 x g for 10 min to remove nuclei.  From the supernatant, mitochondria were pelleted down at 12,000 x g for 15 min.  The crude mitochondria were then incubated with hypotonic buffer on ice for 30 min, whereupon the same volume of hypertonic buffer was added to stop the swelling.  After the centrifugation, samples were treated with RNase A (2 mg/mL) (Life technologies) to remove nuclear and cytosolic RNAs, as previously described (Mercer et al., 2011).  RNase A activity was neutralized by addition of proteinase K (PK; 50 μg/mL).  RNaseOUT (Invitrogen) was used to completely inhibit RNase A activity before RNA isolation.  Mitoplast pellet was obtained by centrifugation at 12,000 x g and treated with RNase A again as described above.  Purified mitoplast pellets were incubted in mitochondria solubilizing buffer at 65 ºC for 5 min.  RNA was isolated using TRIzol reagent, and treated with RNase-free DNase I (Roche) for 1 h at 37 ºC.  RNA was then extracted by acidic phenol (Ambion). RNASeq library was prepared by using TruSeq™ RNA Sample Preparation V2 Kit (Illumina, Cat #: RS-122-2001, RS-122-2002). Poly-A RNA was first purified from 100 ng high quality total RNAs, fragmented to about 150-200 nucleotides in length; then go for 1st-strand and 2nd-strand cDNA synthesis, end repair to remove remove 3’ end overhangs and fill in 5’ overhangs; next an “A” base is added to the 3’ end for adaptor ligation and PCR enrichment (libraries); finally quantify library and check the library quality on Bioanalyzer using DNA 1000 chips. High sensitivity chips are used for further quality control and ensure adequate concentration and optimal fragment size.