After A-tailing using exo-Klenow fragment (New England Biolabs, Ipswich, Massachusetts, NEB M0212L), barcoded adaptors were ligated with Epicentre Fast-Link DNA Ligation Kit (Epicentre LK6201H).
The sheared DNA was end-repaired with Epicentre End-It| End-Repair Kit and size selected for inserts from 2 to 4 kilobases to produce libraries with average insert sizes of 2 Kb, 3 Kb and 3.5 Kb.
Catalonia also celebrates Carnival with gusto, with the epicentre at Sitges – a pretty resort south-west of Barcelona, with a big gay community.
For total mRNA-sequencing libraries, poly(A) + selected RNA was decapped with Tobacco Acid Pyrophosphatase (Epicentre) and fragmented with RNaseIII (ABI).
DNA was extracted with 25:24:1 phenol chloroform:isoamyl alcohol, treated with Riboshredder (Epicentre) for 30 minutes at 37°C, and precipitated with isopropanol.
The embedded DNA was partially digested with Sau3AI and extracted with Gelase (Epicentre, Madison, WI, USA) and dephosphorylated (Antarctic Phosphatase, New England Biolabs).
After isolating total RNA from stable transgenic worms carrying the un-mutated PB plasmid, we digested the RNA with DNase I, dephosphorylated with FastAP™ Thermosensitive Alkaline Phosphatase (Fermentas), decapped with Tobacco Acid Pyrophosphatase (Epicentre), and then ligated to a 5' RACE adapter.
Recent displacement is observed at the Kandern fault, a prolongation of the Vorwald fault, e.g. by the Schopfheim earthquake on 5 May 2009 with a magnitude of M L = 4.5 with an epicentre located about 5 km to the SW of Bürchau (Bundesanstalt für Geowissenschaften und Rohstoffe BGR 2009).
rRNA was removed from 100 µg total RNA by annealing rRNA specific primers, extending with MMLV Reverse Transcriptase (Epicentre Technologies) and digesting the RNA-DNA hybrids with RNAse H (Epicentre Technologies).
It suspended test drilling in June 2011, though, following two earthquakes in the area in April and May of that year, one with a magnitude of 2.3 and the other 1.4, with the epicentre thought to be about 500 metres away from a well in Weeton, Lancashire.
The RNA for libraries 1-11 was enriched with RiboMinus (Ambion®;) while RNA for libraries 12-15 was enriched with RiboZero (Epicentre).
