Template swiching cDNA library construction

Primer sequences

Primers for first strand synthesis:

TS oligo (SfiI site bold) 5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCrGrGrG-3' (rG = ribonucleotide G);

iso3TS oligo (SfiI site bold) 5'-iCiGiCAAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCrGrGrG-3' (iC = iso-dC, iG = iso-dG);

Oligo-dT primer (SfiI site bold) 5'-TAGAGGCCGAGGCGGCCGACATGTTTTGTTTTTTTTTCTTTTTTTTTTVN-3'.

Primers for LD-PCR: iso3TS oligo as 5'PCR primer; 3'PCR primer: 5'-TAGAGGCCGAGGCGGCCGAC-3'.

First strand cDNA synthesis

First strand cDNA synthesis was performed in 106 μl reactions assembled in the following manner. For each sample, the following components were placed in a 200 μl PCR tube on ice: 52.5 μl RNA sample, 8.4 μl oligo dT primer (10 μM), 8.4 μl TS oligo or iso3TS oligo (10 μM), 69.3 μl total volume. RNA and primer mixes were denatured at 65°C for 2 min and then removed to ice. The following reagents were then added to each reaction tube: 20 μl 5× first strand buffer (Powerscript [Clontech] RT buffer: 250 mM Tris (pH 8.3), 30 mM MgCl2, 375 mM KCl, 20 mM DTT), 2 μl DTT (100 mM), 10 μl 50× dNTP (10 mM), 5 μl SuperScript II (Invitrogen, Carlsbad, CA). First strand reactions were incubated at 42°C for 2 hr.

A cleanup step was performed for first strand cDNA samples using Machery-Nagel NucleoSpin Extract II kits following the manufacturer's protocol and the total recovered elution volume was 80-85 μl per sample.

cDNA amplification by Long-Distance PCR (LD PCR)

For PCR amplification of full length cDNAs, 80 μl of cleaned-up first strand cDNA was used as template in 100 μl PCR reactions including the following reagents: 1× Advantage 2 PCR Buffer (Clontech), 0.2 mM dNTPs, 0.12 μM 5'PCR primer (iso3TS oligo), 0.12 μM 3'PCR primer, and 1× Advantage 2 Polymerase Mix. PCR amplifications were performed using an MJ Research Dyad (MJ Research, Waltham, MA, USA) with the following program parameters: an initial hold at 95°C for 1 min, followed by 27 cycles of 95°C for 5 s, 65°C for 5 s, and 68°C for 6 min. After completion of a PCR reaction, a 5 μl aliquot of each sample was removed and run on a 1.1% agarose/TAE gel containing 0.5 μg/ml ethidium bromide alongside a DNA MW marker such as the Fermentas Mass Ruler (Fermentas, Glen Burnie, MA, USA).

LD PCR reaction clean up

LD PCR amplified cDNA samples were cleaned up using NucleoSpin II kits following the manufacturer's protocol and the total combined eluate was approximately 32 μl.

SfiI digestion of cDNA

SfiI digestion was performed in 40.5 μl total volumes containing 32 μl cDNA, 4.05 μl 10× SfiI buffer 4.05 μl SfiI enzyme, and 0.4 μl 100× BSA. Digestions were incubated at 50°C for 2 hrs before proceeding to cDNA size fractionation. SfiI buffer and enzyme were obtained from NEB (Ipswich, MA, USA).

cDNA size fractionation by gel electrophoresis

cDNA was fractionated by molecular weight using preparative DNA gel electrophoresis after SfiI digestion. Digested cDNA samples were loaded (using a loading buffer containing bromophenol blue) on a 2% low melt agarose (NuSieve GTG, Cambrex, Rockland, ME, USA) gel containing 0.5 μg/ml ethidium bromide. Electrophoresis was performed at a low voltage setting (5-8 V/cm) until the bromophenol blue dye had migrated approximately 4 cm. Gels were photographed and the lanes excised from the 400 bp molecular weight marker up to the sample well. DNA was isolated from the gel slice using a Qiagen MinElute kit (Valencia, CA, USA) and 6 sample volumes of buffer QG. Elution was performed using 10 μl of elution buffer. Ligations into pDNR-LIB (Clontech) were performed immediately following cDNA size fractionation and elution according to the method specified in the SMART cDNA library construction manual. Sequencing of gland-derived samples was accomplished using traditional Sanger sequencing of individual clones. The digested, fractionated cDNA samples from rhizome tips and elongation zones were purified using QIAquick PCR purification kit (Qiagen). 5~10 μg purified cDNA from each rhizome sample was used in construction of a 454 Genome Sequencer FLX [21-23] fragment library using Titanium series reagents http://www.454.com followed by sequencing on a GS FLX instrument.