Cloneminer cdna library construction kit

cDNA libraries were constructed as reported in Plett et al., [53 (link), 54 (link)]. Briefly, total RNA (500 μg per sample) was prepared from L. bicolor fruiting bodies (FB), free-living mycelium (FLM) of L. bicolor (strain S238 N) grown in P5 liquid Pachlewski medium for 3 weeks, L. bicolor mycorrhiza root tips and non mycorrhiza roots tips of Populus trichocarpa during a time course of symbiosis development (2, 4, 6 and 12 weeks). RNA extraction was performed using the RNeasy Plant Mini Kit (Qiagen) (for mycorrhiza and non-mycorrhiza root tips, buffer RLC containing 20 mg ml⁻¹ of PEG 8000 was used), followed by a DNase I treatment. Total mRNA were purified using Oligotex columns (Qiagen) and used to build FB + FLM and ECM + Roots cDNA libraries with the CloneMiner cDNA Library Construction Kit (Invitrogen), starting from 2 μg and 500 ng of purified total RNA, respectivley. Entry cDNA libraries were then transferred to the yeast-expressible pDEST32 vector using the Gateway system (ProQuest Two-Hybrid System kit, Invitrogen).

Given that SR3 has demonstrated improved levels of tolerance to both salinity and drought, we proposed to compare the sequence variation of ESTs that are produced under the control and after exposure to saline and osmotic stresses to gain the association between genetic variation and abiotic stress tolerance. Three-leaf-stage seedlings of both cv. SR3 and cv. JN177 were exposed to half strength Hoagland’s liquid medium containing either 200 mM NaCl for 0, 0.5, 1, 12, 24, 48 or 72 h, or to 18 % PEG for 0, 0.5, 1, 12, 24 or 48 h. RNA was extracted from both roots and leaves using the Trizol reagent (Invitrogen, USA), and purified using an OligotexTM-dT₃₀ mRNA Purification Kit (TaKaRa, Japan). Equimolar amounts of the various RNA samples were mixed to obtain both an SR3 and a JN177 mRNA pool, which formed the template for the construction of two cDNA libraries using a CloneMiner™ cDNA Library Construction Kit (Invitrogen, USA), according to the manufacturer’s protocol. Given that the genetic variation induced by asymmetric somatic hybridization was remarkably higher than that induced by other mutagenic factors protoplast formation, UV radiation, callus induction and plant regeneration [31 (link), 32 (link)], other cDNA libraries reflecting the genetic variation of these mutagenic factors were not constructed.

Trepomonas sp. PC1 was isolated from marine sediment near Peggy’s Cove, Nova Scotia, Canada, and grown in the lab on a mixed culture of bacteria. Total RNA was collected from several independently grown cultures in 50 mL Falcon tubes. Messenger RNA was purified from total RNA using the Poly(A)Purist™ MAG system (Ambion). Directed and size-fractionated cDNA libraries were made using the CloneMiner™ cDNA Library Construction Kit (Invitrogen), which were then sequenced with Sanger technology. The rest of the mRNAs were sequenced with Illumina Genome Analyzer IIx instrument, producing 41 million 100 bp long single reads. Raw RNA sequence reads were deposited at NCBI Sequence Read Archive (SRA) under accession number SRR2079337. The isolate is no longer in culture.