TITLE: Library Adapter Preparation for Dual-Index Double Stranded DNA  Illumina Sequencing
AUTHORS: Franziska Aron, Guido Brandt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Adapter preparation protocol for doubble-stranded ancient DNA libraries for Illumina Next-Generation-Sequencing, based on Meyer and Kircher </span><span style = "font-style:italic;">et al.</span><span> (2010) </span><span style = "font-style:italic;">Cold Spring Harb. Protoc.</span><span> (doi: </span><a href="http://dx.doi.org/10.1101/pdb.prot5448" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">10.1101/pdb.prot5448</span></a><span>). </span></div><div class = "text-block">The protocol describes the production of an adapter mix for 2000 libraries.</div><div class = "text-block"><span>This protocol is used in conjunction with Meyer and Kircher-based </span><span style = "font-weight:bold;">ancient DNA libraries </span><span>and is described accordingly (see library construction like: Non-UDG treated double-stranded ancient DNA library preparation for Illumina sequencing).</span></div></div>

[STEPS]
?. [Adapter Preparation (Library Preparation Room)]
Prepare P5 adapter ()Use one tube of a 0.2 ml PCR strip to set up the P5 adapter mix.  ABCD1ReagentStock concentrationFinal concentration1× Volume [µl]2Oligo Hybridization Buffer10 ×1 ×103IS1_adapter.P5500 µM200 µM404IS3_adapter.P5+P7500 µM200 µM405UV HPLC-water106Total100Gently pipette up anddown to mix. Split the  reaction into two tubes of the 0.2ml PCR strip with  .
[per reaction]
ABCD1ReagentStock concentrationFinal concentration1× Volume [µl]2Oligo Hybridization Buffer10 ×1 ×103IS1_adapter.P5500 µM200 µM404IS3_adapter.P5+P7500 µM200 µM405UV HPLC-water106Total100
100 µl
[each]
?. [Adapter Preparation (Library Preparation Room)]
Prepare P7 adapter ()Use one tube of a 0.2 ml PCR strip to set up the P7 adapter mix.  ABCD1ReagentStock concentrationFinal concentration1× Volume [µl]2Oligo Hybridization Buffer10 ×1 ×103IS2_adapter.P7500 µM200 µM404IS3_adapter.P5+P7500 µM200 µM405UV HPLC-water106Total100Split the  reaction into two tubes of the 0.2ml PCR strip with  .
[per reaction]
ABCD1ReagentStock concentrationFinal concentration1× Volume [µl]2Oligo Hybridization Buffer10 ×1 ×103IS2_adapter.P7500 µM200 µM404IS3_adapter.P5+P7500 µM200 µM405UV HPLC-water106Total100
100 µl
[each]
?. [Adapter Preparation (Library Preparation Room)]
Prepare ready-to-use adapter mix.
?. [Hybridization buffer preparation (Buffer Preparation Room)]
Prepare oligo hybridization buffer (10× concentration, )  ABCD1ReagentStock concentration [M]Final Concentration [M]1× Volume [µl]2NaCl50.51003Tris-HCl10.01104EDTA0.50.00125UV HPLC-water8886Total1000
[ for 50 reactions]
ABCD1ReagentStock concentration [M]Final Concentration [M]1× Volume [µl]2NaCl50.51003Tris-HCl10.01104EDTA0.50.00125UV HPLC-water8886Total1000
The oligo hybridization buffer can be stored at room temperature for up to one year.
?. [Adapter Preparation (Library Preparation Room)]
Combine one P5 adapter reaction with one P7 adapter reaction in one tube, mix thoroughly by flicking the tubes with a finger, spin down briefly.Repeat with the second P5 and P7 adpater reaction.
In the end you should have two 0.2 ml tubes with a total of   volume, each containing   of the P5 adapter and   of the P7 adapter.
?. [Adapter Preparation (Library Preparation Room)]
Incubate both reactions in a thermocyler with a heated lid at  for , followed by a cool down ramp from  to  at a rate of .
95 °C
95 °C
12 °C
[per sec]
?. [Combine and Dilute]
Add of UV HPLC-water to dilute the double-stranded library adapter mix to  and aliquot the dilution in 10 × 1.5 ml LoBind tubes, each containing . Briefly vortex and spin down before freezing.
1800 µl
200 µl
?. [Combine and Dilute]
Combine both reactions into a 2 ml tube to obtain a ready-to-use double-stranded library adapter mix with .
[adapter (each)]
?. [Hybridization buffer preparation (Buffer Preparation Room)]
Irradiate the buffer with UV for  without the lid or with an open lid.
?. [Combine and Dilute]
Store the adapter mix at
-20 °C
Each aliquot contains sufficient double-stranded library adapter mix for 200 reactions (overall 2000 libraries). The adapter mix aliquot can be thawed and re-frozen with no detriment to the reagent quality.