SAMN00168377
External Id
LIBEST_025378
External Id
University of British Columbia
INSDC center name
2009-11-17T00:00:00Z
INSDC first public
2023-10-26T21:06:24.581Z
INSDC last update
live
INSDC status
Generic
NCBI submission model
Generic.1.0
NCBI submission package
ERS16610518
SRA accession
Grosmannia clavigera (Ophiostoma clavigerum) isolate SLKW1407 (UAMH 11150) spores for inoculating petri-plates were generated from mycelial cultures grown seven days on 1percent malt extract agar (MEA, Oxoid; 10 cm diam. petri plates; harvested with 5 ml autoclaved dH2O/plate). After adjusting to 5.0x10^5/ml, spores were aliquoted in 100 ul of dH20 and spread evenly onto fifty 1percent MEA (Difco) 10 cm petri plates. After seven days, spores were harvested using autoclaved dH2O (5ml/plate) and filtered through a BD Falcon cell strainer (40 um) to remove mycelial debris. After harvesting, the spore suspension was centrifuged at 3,000 rpm for 15 minutes. Most of the water was removed, samples were pooled, then spores were transferred to 1.5 ml microtubes. Samples were centrifuged for 2 minutes at 10,000 rpm and the remaining H2O was removed. An un-normalized, full-length enriched, directionally cloned library was prepared with the Creator SMART cDNA Library Construction Kit (Clontech, Mountain View, CA, USA) and the TRIMMER-DIRECT cDNA Normalization Kit (Evrogen, Moscow, Russia). The Evrogen kit was used for some of the protocols and for the CDS-3M primer but this is not a normalized library. Total RNA (50 micrograms) was extracted using the following methodology. Spores were suspended in an appropriate volume of buffer RLT (RNeasy, Qiagen) and disrupted using a fastprep homogenizer with lysing matrix A (MPBiomedicals). After homogenizing, debris and lysing matrix were separated from supernatant by centrifuging 1 minute at 14,000 rpm and then the supernatant was processed using the Qiagen RNeasy Midi Kit following the manufacturers protocol. mRNA (276 nanograms) was purified from total RNA (32 micrograms) using the Oligotex mRNA Kit (Qiagen). First strand cDNA was prepared from 69 nanograms of mRNA, SuperScript III reverse transcriptase (Invitrogen), CDS-3M primer (Evrogen), and the SMART IV Oligonucleotide (Clontech). Second strand cDNA was prepared by LD-PCR with Phusion Hot Start DNA Polymerase (Finnzymes, Espoo, Finland), digested with SfiI, size fractionated, and cDNA larger than approx. 500 bp was ligated into SfiI-digested pDNR-LIB (Clontech). The ligation mixture was transformed into ElectroMAX DH10B T1 Phage-Resistant electro-competent cells (Invitrogen). Titre of the primary library (1.8 mL) was approx. 1.3x10^6 cfu/mL and was not amplified.
description
SLKW1407 (UAMH 11150)
isolate
E. coli DH10B
lab-host
Leptographium clavigerum
http://purl.obolibrary.org/obo/NCBITaxon_226899
organism
Sfi I
re_1
Sfi I
re_2
Spore
tissue-type
Ocl08-Kw1407-spore-Unnorm (OCL08)
title
Plasmid
http://purl.obolibrary.org/obo/SO_0000155
v_type
pDNR-LIB
vector