RNA/transcriptome
Focusing on only the transcribed portion of the genome. High-throughput transcriptome sequencing gives
versatile data, which can simultaneously be analysed to provide insight into the level of gene expression, the
structure of genomic loci, and sequence variation present at loci (e.g. SNPs, gene tree estimation).
Procedure:
Samples are sent to the SciLifeLab and you need to place an order in the SciLifeLab Web Portal.
From SciLifeLab:
“When you have filled in an order in the web portal and we have agreed on a suitable setup and cost for
the project you will get a Contract to sign, and as soon as we have the signed contract and detailed
information of your quality controlled samples you may send us your samples.
Before you send us anything, please read this document and fill out Sample Information
(20143) or Library Information (20197) that you received together with the Contract and send it by
email to genomics_orders@scilifelab.se. If you have not received the appropriate form, please request
one by sending a message to genomics_orders@scilifelab.se.”
We have previously had contact with Mattias Ormestad who is a project coordinator at SciLifeLab, so
might be a good idea to stick with the same person for your project.
RNA extraction
RNA quality control and shipping of samples After extracting your RNA, use the Agilent RNA ScreenTape
system to check the quality of your RNA, this to ensure that high quality starting material is used for
the library preparation. You run your samples on the Agilent 2200 TapeStation (room 3304B). The aim
is to have samples with

High RNA concentration. Ideally between 50-250 ng/µL.

High RINe value. RINe values indicate the RNA quality and range from 1-10. Ideally
your samples should have a minimum of 8.

Nice peaks on the chromatograms
If your samples have a much higher concentration than the advised 50-250 ng/µL you need to dilute
the samples before sending for library preparation, but do not worry too much if RINe values are too
low or if the chromatograms are not perfect.
1)
Online protocol READ – important additional notes!!
2)
Sample preparation – room 3122 “Pre pcr-room”
a.
Place R6K Sample Buffer in room temperature 30 min and vortex prior to use - the buffer
contains DMSO, which solidifies when cold. DMSO also easily penetrates trough skin –
USE GLOVES!
b.
Work in the fume hood and mix 4µL R6K Sample Buffer with 1µL of your RNA sample.
Sample Buffer mixed with RNA sample should be kept on ice during sample preparation
and after sample denaturation.
c.
It is of course always good to include a negative sample (Sample Buffer + 1µL milliQ
water)
d.
You need to run an even number of samples, so if necessary add a negative sample or a
sample with only water.
e.
Sample denaturation:
f.
3)
4)
5)
6)
i.
Heat the samples to 72°C for 3 min.
ii.
Place samples on ice for 2 min.
iii.
Briefly centrifuge the samples to collect the contents in the base of the tubes.
When you are finished with the sample preparation – clean fume hood with water (spray
can above fume hood) and close the lid. Start the UV timer and run for 30 min.
Prepare TapeStation – room 3304 B
a.
Bring your samples to the TapeStation (remember to keep them on ice!) and also bring
the R6K ScreenTape from the fridge in room 3122.
b.
Turn on the computer – user: admin, password: 2200.
c.
Turn on the Agilent 2200 TapeStation.
d.
Load the R6K ScreenTape and the special loading tips (they are in the shelf above the
machine). Make sure there are no bubbles in the separation channels by “flicking” the
ScreenTape prior to loading.
e.
Start the TapeStation software (TapeStation Controller) – it is located on the Desktop on
the computer.
Sample Analysis
a.
Load your samples in the machine and remove the lids/caps. !!!THE MACHINE WILL
BREAK IF YOU DO NOT REMOVE THE LIDS!!!
b.
Select the required samples on the TapeStation Controller software.
c.
Click START and specify a file to which the results will be saved.
d.
It takes about 1 min for each sample to run.
Checking results
a.
As the analysis is finished you can open your result file in the TapeStation Analysis
software. Here you can see the gel image, sample information and chromatograms. As
stated above you aim for high RNA concentrations, RINe value >8 and chromatograms
with two distinguishable peaks at about 2000 and 4000 nt.
b.
If any sample has a too high RNA concentration, you need to dilute the sample and run
the TapeStation analysis anew. Previous experience with dilutions show that you need to
dilute more than what you initially think. It might be wise to do a couple of different
dilutions at once to ensure that you get the required RNA concentration.
Preparing and shipping samples
a.
We send the samples to SciLifeLab. For more information about the sample requirements
for genomic projects please follow this link. The reports from the quality control (RNA
concentrations, RINe value and chromatograms) should be sent to your contact person
before sending the samples. Send the samples in the beginning of the week to avoid
packages being stuck someplace over the weekend.
b.
You need to send the samples in special 96-well barcoded colored plates. These plates are
provided by the SciLifeLab and should be sent to us as the order has been placed on their
web portal, but make sure that they really send it.
c.
Transfer between 20-100 µL of sample into the plates and use the special seal that comes
along with the plates.
d.
RNA samples must be sent on dry ice. The dry ice is ordered from AGA. Contact Vivian
Alden to make the order.
e.
Place your samples in the dry ice package and seal properly with tape.
f.
We use TNT for shipping the samples. Contact Sven Toresson who will help you with the
order. You need to order before 15:00 to ensure that the samples will be picked up the
same day.
g.
We have previously sent our samples to Mattias Ormestad, but this probably depends on
which contact person you have. But this is his address (it is important to include a phone
number):
Mattias Ormestad
SciLifeLab
KISP
Tomebodavägen 23B
17165 Solna
SWEDEN
Phone: 070-7127871 or 08-52481435
h.
Place the package on the delivery bridge “lastbryggan” on the table marked “Avgående
paket” on the second floor.
i.
OK! Now samples are on the way and you can just keep your fingers crossed that you
will get nice results back from SciLifeLab!