Illumina HiSeq 2000 sequencing; XRNAX_RiboZero_C74M6ACXX_lane1
Protocols :  Extraction Protocol: Up to 100 million cells were lysed in 8 ml TRI reagent by pipetting up and down. Cell clumps were disintegrated by flushing the lysate repeatedly against the wall of the tube. Lysis was further facilitated by incubation on a rotating wheel for 5 minutes at room temperature. Lysates were combined with 1.6 ml chloroform, mixed by inversion and incubated for 5 minutes at room temperature. Tubes were spun down with 7000 g for 10 minutes at 4 C. The aqueous phase was removed and the interphase transferred to a 2 ml tube. The interphase was gently washed twice with 1 ml low SDS buffer (Tris-Cl 50 mM, EDTA 1 mM, SDS 0.1 %), flushing protein off the walls of the tube while retaining the integrity of the interphase flakes. Flakes were spun down with 5000 g for 2 min at room temperature and the supernatant discarded. After the washing, flakes were disintegrated by pipetting into 1 ml of low SDS buffer. The disintergrated interphase was spun down with 5000 g for 2 min at room temperature and the supernatant saved as interphase eluate 1. Disintegration of the interphase was repeated with another 1 ml of low SDS buffer, then twice with 1 ml of high SDS buffer (Tris-Cl 50 mM, EDTA 1 mM, SDS 0.5 %) each time yielding approx. 1 ml of interphase eluates. NaCl was added to a final concentration of 300 mM to each of the 4 interphase eluates, along with 1 microl GlycoBlue and 1 ml isopropanol before mixing by inversion. Samples were spun down for 15 min with 18000 g at -10 C. The supernatants were discarded and pellets from all four elutes were combined in 2 ml of 70 % ethanol. The combined sample was again centrifuged for 1 min with 18000 g at room temperature, supernatant discarded and all residual ethanol removed. The pellet was taken up in 1.8 ml of nuclease-free water and detached from the wall of the tube with a pipette tip. The pellet was allowed to swell for 1 hour on ice with occasional mixing by inversion and eventually dissolved by pipetting. 200 microl NEB DNase I buffer 10 x was added along with 2 microl RNasin Plus, 100 microl NEB DNase and incubated for 60 minutes at 37 C and 700 rpm shaking. Subsequently, the sample was isopropanol precipitated as described above without further addition of GlycoBlue. Pellets were taken up in 1000 microl nuclease-free water and dissolved by pipetting. For a detailed, photo-documented version of the XRNAX protocol visit www.XRNAX.com/theprotocol.  Library Protocol: Untranded RNA-seq library preparation. RRNA was NOT depleted using RiboZero (Illumina).
Factor Value[Growth Media]
DMEM
Factor Value[Treatment]
UV254