Generation of Solexa sequencing libraries

3.2.4.1 Gel purification of RNA

22-60 µg of RNA were separated on a 20% Sequagel Acrylamide-Urea gel (National Diagnostics; Atlanta, USA) at constant 250V for 45 to 60 min. 5 µl microRNA marker (New England Biolabs; Ipswich, USA) consisting of 17, 21 and 25 nt bands, was used as size control. After staining the gel in 1x SybrGold (Invitrogen; Karlsruhe, Germany) for 5 min, the bands of small RNAs were excised corresponding to the desired size from 17 to 30 nt. An 0.5 ml Eppendorf tube pierced with a .22 gauge needle was used to shredder the gel slice (full speed, 5 min). 500 µl of Solexa elution buffer (0.4M NaCl, 0.5% SDS, 50 mM Tris- HCl pH 8) and 1 µl Proteinase K was pipetted into the shred and shaken for at least 2 hours at 65°C to elute the RNA. The gel slices were eliminated by centrifuging (full speed, 2 min) through empty spin column

(MoBiTec; Göttingen, Germany). The eluted RNA was supplemented with 30 µg glycogen and 400 µl Phenol/Chloroform/IAA, pH 4.5-5 (Roth; Karlsruhe, Germany) and centrifuged full speed for 30 min at 4°C. The supernatant was transferred, precipitated with an equal volume of isopropanol, mixed well, incubated for 15 min at RT and centrifuged full speed for 20 min at RT. The supernatant was removed and washed twice with 150 µl of 70% ethanol. Finally RNA was dried for 1 min with the lid closed and resuspended in 8 µl RNase free water.

3.2.4.2 Linker ligation at 3’ end of RNA

For linker ligation at 3’ end of RNA, the reaction mix was as follows: Gel purified RNA (resuspended in water) 6 µl ATP-free T4 RNA ligase buffer (10x) 1 µl

DMSO 1 µl

Modban oligo (50 µM) 1 µl

Mutant RNA ligase (self-made) 1 µl

After incubation for 15 min at 37°C, the ligation reaction was mixed with 10 µl 2x formamide loading dye and inactivated at 95°C for 5 min.

The truncated T4 RNA ligase was taken from our own laboratory stock (see Methods 3.2.6). The corresponding ATP-free T4 RNA ligase buffer (500 mM Tris-HCl pH 7.5-7.6, 100 mM MgCl2, 100 mM DTT,

600 µg/mL BSA) was aquired from New England Biolabs (Ipswich, USA).

3.2.4.3 Gel purification of ligated RNA product after 3’ ligation

The ligated RNA products were separated on a 15% Acrylamide-Urea gel (National Diagnostics; Atlanta, USA) at 250 V for 45 to 60 min. After staining of the gel in 1x SybrGold, miRNA marker and 50 bp ladder (New England Biolabs; Ipswich, USA) were used as size control to excise the band corresponding to the desired size of small RNA of 36 to 41 nt. The RNA elution from the gel as well as the RNA precipitation and the final dissolving in water were carried out as explained above in 3.2.4.1.

3.2.4.4 Linker ligation at 5’ end of RNA

For linker ligation at 5’ end of RNA, the reaction mix was as follows: Ligated product (resuspended in water) 6 µl

T4 RNA ligase buffer (10x) 1 µl

DMSO 1 µl

Solexa linker (50 µM) 1 µl

T4 RNA ligase 1 µl

After the incubation for 1 hour at 37°C, the T4 RNA ligase was inactivated at 95°C for 5 min. T4 RNA ligase and the appropriate buffer were acquired from Life Technologies; Carlsbad, USA.

3.2.4.5 Gel purification of ligated RNA product after 5’ ligation

The following gel purification step of RNA after 5’ ligation was used for the first Solexa sequencing run (cell cycle: G1, early S, late S and G2) while it was skipped for the later approaches.

The ligated RNA products were separated on a 10% Acrylamide-Urea gel (National Diagnostics; Atlanta, USA) at 250 V for 45 to 60 min. After staining of gel in 1x SybrGold, 50 bp ladder (New England Biolabs; Ipswich, USA) were used as size control to excise the band corresponding to the desired size of small RNA around 100 nt length. The RNA elution from the gel as well as the RNA precipitation and the final dissolving in water were carried out as explained above in 3.2.4.1.

3.2.4.6 Reverse transcription

The reverse transcription of ligated RNA is adapted to the Superscript II Reverse Transcriptase protocol (Invitrogen; Karlsruhe, Germany):

Ligated RNA product 9 µl

BanOne primer (5 µM) 2 µl

After incubation at 95°C for 2 min, the mix is cooled on ice for 2 min and centrifuged briefly at RT. The following components are added:

First strand buffer (5x) 4 µl

DTT (0.1 M) 2 µl

dNTP Mix (10 mM each) 1 µl

The resulting reaction was mixed gently, split into two tubes comprising 9 µl each and incubated at 42°C for 3 min. After addition of 1 µl Superscript II RT (Invitrogen) to the sample or 1 µl H2O as negative control, the

content of the tubes were incubated at 42°C for 30 min. The reaction was inactivated by heating at 95°C for 5 min and cDNA stored at -20°C.

3.2.4.7 PCR amplification of cDNA

cDNA (+RT) or control (−RT) 5 µl

PCR buffer (5x; Mg2+ final conc.: 2.5 mM) 20 µl

dNTP Mix (10 mM each) 2 µl

5’-Primer - Solexa (10 µM) 1 µl

3’-Primer - PCR BamHI/Pvu/Xba/Cla (10 µM) 1 µl Phusion Hot Start DNA Polymerase 1 µl

H2O 70 µl

Thermocycler protocol:

OLD PROTOCOL NEW PROTOCOL

2 min 94°C initial denaturation 2 min 94°C

--- 5 cycles: 15 sec 94°C denaturation --- 30 sec 54°C annealing --- 30 sec 72°C extension --- --- 17 cycles: 23 cycles:

15 sec 94°C denaturation 15 sec 94°C

30 sec 60°C annealing 30 sec 60°C

30 sec 72°C extension 30 sec 72°C

---

2 min 72°C final extension 2 min 72°C

storage at 4°C storage at 4°C

PCR products were separated by agarose gel electorophoresis (2% agarose gel), excised, purified by QIAGEN Gel Extraction Kit and finally eluted with 30 µl Elution buffer.

Furthermore different primers were used for old or new protocol as the bar codes were introduced at different steps during 5’ ligation or PCR reaction, respectively (see Figure 3.2). The primer sequences can be found in chapter 3.1.11.9.

Figure 3.7 Overview of primer use for multiplexing experiment in generation of small RNA libraries.

3.2.4.8 Ligation of purified cDNA with pJET 1.2/blunt

Ligation of purified cDNA with pJET1.2/blunt was performed according to the CloneJETTM _{PCR Cloning Kit}

protocol.

PCR product 4 µl

pJET1.2/blunt cloning vector (50 ng/µl) 1 µl

Reaction buffer (2x) 10 µl

T4 DNA ligase 1 µl

H2O 4 µl

The incubation time was extended up to 30 min at RT to obtain the maximal number of transformants.

3.2.4.9 Bacterial transformation

Transformation of competent bacteria was carried out by standard heat shock procedures. Briefly, 50 μl XL2-blue CaCl2-competent cells were thawed on ice. 5-8 μl of ligation sample were added and the mixture

was incubated on ice for 30 min, subjected to a 2 min heat shock at 42°C and returned to ice for 1 min. 1 ml SOC-medium was added and cells were allowed to grow for 1 h in at 37°C shaking incubator. Afterwards

cells were centrifuged for 30 sec at full speed in a table top centrifuge. The supernatant was removed and the resuspended cell pellet was streaked out on agarose plates with Ampicillin (Amp) antibiotic for selection of transformants.

3.2.4.10Test for correct transformants by colony-PCR

Individual colonies were tested for their insert by colony-PCR with a primer pair contained in the CloneJETTM

PCR Cloning Kit (pJET1.2 fw: 5’-cgactcactatagggagagcggc-3’; pJET1.2 rev: 5’-aagaacatcgattttccatggcag-3’). A following standard PCR reaction mix was inoculated with a single colony, which was subsequently streaked onto a fresh plate and labeled for later recognition. Standard amplification was carried out with 10 min initial denaturing for cell lysis of bacteria.

Taq buffer (+KCl, -MgCl2) (10x) 2 µl MgCl2 (50 mM) 0.6 µl dNTP Mix (10 mM each) 0.4 µl pJET 1.2_s (10 µM) 0.4 µl pJET 1.2_as (10 µM) 0.4 µl Taq polymerase 0.1 µl H2O 16.1 µl Thermocycler protocol:

10 min 95°C initial denaturation

--- 35 cycles: 30 sec 94°C denaturation 30 sec 55°C annealing 30 sec 72°C extension --- storage at 4°C

PCR products were separated by agarose gel electorophoresis, excised and purified by QIAGEN PCR Purification Kit.

3.2.4.11DNA sequencing

The sequences of the obtained inserts were investigated by sequencing (Eurofins MWG, Ebersberg, Germany). Further analysis of the sequences and alignments were performed with ApE (A plasmid Editor; http://biologylabs.utah.edu/jorgensen/wayned/ape/) and the BLAST function of http://flybase.org.

3.2.4.12Bioinformatic analysis of deep sequencing data

Solexa sequencing for total RNA libraries was carried out at Fasteris (Plan-Les-Ouates, Switzerland) while the sequencing of libraries consisting of beta-eliminated RNAs was performed at the Gene Center (Munich, Germany).

The sequences were mapped onto the target sequences using BOWTIE (http://bowtie-bio.sourceforge.net/) with the option –n0 to force selection of only perfectly matching sequences. Pre-processing of sequences and analysis of the BOWTIE output files were done using PERL scripts.