title: pool of 20 females of Ixodes ricinus collected in natural population in France
organism: Ixodes ricinus
description: Tick collections and DNA extraction :
   The DNA libraries to be used to detect suitable SNPs were constructed by sampling two I. ricinus populations. Only adult females (the life cycle stage with the largest amount of DNA) were used.
   The M population corresponds to 20 non-engorged females that were collected in spring 2011 (and kept alive at 4°C in the lab until DNA extraction) as they were questing for a host on vegetation in north-west France ('Malville'; 47° 21' 30.10" N, 1° 51' 41.59"W). Each individual was extracted and the DNA concentration measured separately. 
   The ticks were frozen in liquid nitrogen and crushed with a pestle in individual tubes. DNA extraction was conducted according to manufacturer instructions using the Nucleospin tissue XS kit (Macherey-Nagel).
   
   Genome reduction and sequencing
   After testing several restriction enzymes, MseI (T ^ AAT; New England Biolabs) was selected because a high concentration of DNA fragments of the target size could be obtained (id est 500-600b, to maximize the efficiency of 454 pyrosequencing relative to reads length) and no discrete band (that would reveal the presence of repeat elements) was observed in the target range of DNA fragments selected. Each sample was digested for 8 hours (2.5U/µg of DNA) according to manufacturer's instructions. The digested DNA was then separated on a 1% agarose gel (4h, 80v). A gel piece of each sample, containing DNA fragments from 500 to 600 bp (according to the 100 pb marker size ladder; Eurobio) was sheared under a UV lamp. The DNA was then extracted and cleaned with the gel clean-up kit (Macherey-Nagel), eluted in 40 µl of EB Buffer (3.33mM Tris, pH 8.5), then quantified using Qubit™ with the dsDNA HS Assay™ kit (Invitrogen). Each sample was then sent to the Biogenouest Genomic platform (Rennes, France) (where the 454 sequencing was conducted). The pool was prepared from an equimolar quantity of DNA from each of the 20 samples respectively, corresponding to 500ng of DNA in 10 μl. 
   This pool was tagged with a unique barcode (multiplex identifier MID), and was then sequenced using the Roche 454 GS FLX and Titanium chemistry.
   
attributes.tag: collection_date
attributes.value: 2011
attributes.tag: geo_loc_name
attributes.value: France: Loire-Atlantique
attributes.tag: dev_stage
attributes.value: adult
attributes.tag: sex
attributes.value: female
attributes.tag: strain
attributes.value: natural population
attributes.tag: isolate
attributes.value: "M"