Garber Lab RNA-seq

protocol
University of Massachusetts Medical School
Program in Bioinformatics and Integrative
Biology
368 Plantation Street
Worcester, Massachusetts 01655

PI: Manuel Garber

RNA isolation.
(RNeasy Plus Mini Kit Qiagen cat # 74134 with additional On-Column DNase Digestion
with the RNase-Free DNase Set Qiagen cat. # 79254)
1) Harvest around 0.5-1 Mln monocyte derived dendritic cells and centrifuge at 500xg for 5 minutes at
RT. Immediately put cell pellets on dry ice and stored frozen at -80C till ready, or resuspend in RLT
Plus buffer (350 µL) prior storage at -80C.
2) When ready for RNA isolation remove samples from -80C and thaw cells pellets or cell lysates on ice.
Resuspend cell pellets in RLT Plus buffer (350 µL).
3) Vortex each samples for 30 s.
4) Transfer the lysate to a gDNA Eliminator spin column placed in a 2 ml collection tube. Centrifuge for
30 s at ≥8000 x g. Discard the column, and save the flow-through.
5) Add 1 volume (350 µl) of 70% freshly prepared ethanol to the flow-through, and mix well by
pipetting. Proceed immediately to the next step.
6) Transfer 700 µl of the sample, including any precipitate, to an RNeasy spin column placed in a 2 ml
collection tube. Close the lid, and centrifuge for 15 s at ≥8000 x g. Discard the flow-through.
7) Add 350µl Buffer RW1 to the RNeasy Mini spin column (in a 2 ml collection tube). Close the lid, and
centrifuge for 15 s at ≥8000 x g. Discard the flow-through.
8) Do the additional DNase treatment: Add 10 µl DNase I stock solution to 70 µl Buffer RDD (RNase-
Free DNase Set, Qiagen cat #79254). Mix by gently inverting the tube, and centrifuge briefly to
collect residual liquid from the sides of the tube. DNase I is especially sensitive to physical
denaturation. Mixing should be only be carried out by gently inverting the tube. Do not vortex!
9) Add the DNase I incubation mix (80 µl) directly to the RNeasy spin column membrane, and place on
the benchtop (20–30°C) for 15 min. Be sure to add the DNase I incubation mix directly to the RNeasy
spin column membrane.
10) Add 350 µl Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at
≥8000 x g. Discard the flow-through.
11) Add 500 µl Buffer RPE to the RNeasy spin column. Close the lid, and centrifuge for 15 s at ≥8000 x g.
Discard the flow-through.
12) Add 500 µl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at
≥8000 x g.
13) Place the RNeasy spin column in a new 2 ml collection tube and centrifuge at full speed for 1 min to
further dry the membrane.
14) Place the RNeasy spin column in a new 1.5 ml collection tube.
15) Add 30–50 µl RNase-free water directly to the spin column membrane. Close the lid, and
centrifuge for 1 min at ≥8000 x g to elute the RNA.
16) Check RNA concentration and quality on a Bioanalyzer (RNA chip standard sensitivity).
17) Keep RNA samples at -80C.

RNA-seq library preparation:

1. RNA-seq libraries were prepared using Ovation Mouse RNA-Seq Library
Systems kit (NuGEN Cat# 0348) using 100ng of total RNA as input