TITLE: No Title
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[DESCRIPTION]
This protocol is an update of the Metatranscriptomics protocol from DeLong lab 2012 which incorporates ScriptSeq v2 RNA library preparation.<br /><br />Part A: RNA extraction, DNase treatment, purification, and quantification.<br />Part B: rRNA subtraction<br />Part C: cDNA synthesis and Illumina library prep

[BEFORE_START]
<br />* &nbsp;Prior to all lab work, wipe down bench surfaces and lab equipment with RNase-Zap (Ambion) to deactivate RNases.<br />* &nbsp;Replace gloves frequently during the procedure.<br />* &nbsp;Confirm the availability of all downstream reagents before proceeding to the next step.

[GUIDELINES]
<strong>General notes:</strong><br /><br />* &nbsp;Prior to all lab work, wipe down bench surfaces and lab equipment with RNase-Zap (Ambion) to deactivate RNases.<br />* &nbsp;Replace gloves frequently during the procedure.<br />* &nbsp;Confirm the availability of all downstream reagents before proceeding to the next step.<br /><br /><strong>Part A: RNA exraction, DNase treatment, purification, and quantification</strong><br />-- for filters in 300&nbsp;&micro;l RNAlater<br /><br /><strong>A1</strong>. RNA extraction<br />--- based on&nbsp;<em>mir</em>Vana<sup>TM</sup> miRNA Isolation kit (Ambion)<br /><br />Preparation:<br /><br />- &nbsp;Add 21 ml of ACS grade 100% ethanol to Wash Solution 1, and mix well.<br />- &nbsp;Add 40 ml of ACS grade 100% ethanol to Wash Solution 2/3, and mix well.<br />- &nbsp;Make sure the Acid-Phenol:Chloroform is not more than 6-months old.<br /><br /><br /><strong>A2</strong>. DNase Treatment (~100&nbsp;&micro;l rxn)<br />---based on Ambion TURBO DNA-free<sup>TM</sup> kit<br /><br />Preparation: set heat block or hyb oven to 37&deg;C<br /><br /><strong>A3</strong>. Concentrating and purifying DNase treated RNA<br />---based on RNeasy MinElute Cleanup Kit (Qiagen)<br /><br />Preparation: add 4 volumes of ethanol (100%) to buffer RPE.<br /><br /><strong>A4</strong>. RNA quantification<br /><br />Depending on your application, the recommended option for RNA quantification is:<br /><br />1. &nbsp;Bioanalyzer (Agilent) using the RNA 6000 Pico total RNA kit: quantitative range = 0.05-5.0 ng/&micro;l (dilute samples 1:10 and 1:100 prior to analysis), allows visualization of RNA size distribution, less accurate, ~1 hr processing time.<br /><br /><br /><strong>Part B: rRNA subtraction<br /><br />B1</strong>. PCR amplification of rRNA genes<br /><strong><br /></strong>This step creates sample-specific amplicon pools using DNA template extracted from samples collected in parallel with those used to generate the total RNA extract. It is important to use DNA template that roughly mirrors the taxonomic composition of the microbial community in the samples to be subtracted. The sample-specific amplicon pools will be used as template for in vitro transcription (using T7 RNA polymerase) to produce anti-sense rRNA probes complimentary to rRNA in the total RNA extract. <br /><br />Primers:<br /><br />Bacteria<br /><br />16S:<br /> <strong>Eub16S_27F</strong>&nbsp; AGAGTTTGATCCTGGCTCAG<br /> <strong>Eub16S_1492R_T7</strong> GCCAGTGAATTGTAATACGACTCACTATAGGGACGGCTACCTTGTTACGACTT<br /> <br />23S:<br /> <strong>Eub23S_189F</strong>&nbsp;&nbsp; GAASTGAAACATCTHAGTA<br /> <strong>Eub23S_2490R_T7</strong>&nbsp; GCCAGTGAATTGTAATACGACTCACTATAGGGCGACATCGAGGTGCCAAAC<br /><br />Archaea<br /><br />16S:<br /><strong>Arch16S_21F</strong>&nbsp; TCCGGTTGATCCYGCCGG<br /><strong>Arch16S_1492_T7&nbsp;</strong> GCCAGTGAATTGTAATACGACTCACTATAGGGGGYYACCTTGTTACGACTT<br /><br />23S: <br /><strong>Arch23S_F</strong>&nbsp; ASAGGGTGAHARYCCCGTA<br /><strong>Arch23S_R_T7&nbsp;</strong> GCCAGTGAATTGTAATACGACTCACTATAGGGCTGTCTCRCGACGGTCTRAACCCA <br /><br />Eukaryotes<br /><br />18S:<br /><strong>Euk18S_1F</strong>&nbsp; ACCTGGTTGATCCTGCCAG<br /><strong>Euk18S_1520R_T7</strong>&nbsp; AATTATAATACGACTCACTATAGATTCYGCAGGTTCACCTAC<br /><br />28S:<br /><strong>Euk28S_26F</strong>&nbsp; ACCCGCYGAAYTTAAGCATA<br /><strong>Euk28S_3126R_T7</strong>&nbsp; AATTATAATACGACTCACTATAGATTCTGRYTTAGAGGCGTTCAG<br /><br />See DeLong et al. 1999 (AEM 65:5554-5563) for design of T7-appended primers and Hunt et al. 2006 (AEM 72:2221-2225) for bacterial 23S primers. Archaeal primers are based on conserved stretches in an alignment of taxa representing major Crenarchaeal and Euryarchaeal divisions. Eukaryotic primers are based on conserved stretches in representative marine taxa. The 5&rsquo; bases upstream of the T7 promoter facilitate RNA polymerase binding. Transcription efficiency increases if the first bases downstream of the promoter are GG in the transcribed sequences (CC in the template strand).<br /><br />Protocol for 50 &micro;l reaction* (using Herculase&reg; II Fusion polymerase, Agilent 600675):<br /> <br />
<table>
<tbody>
<tr>
<td>Template DNA</td>
<td>100 ng</td>
<td>Bact 23S (35 cycles)</td>
<td>All other primers (35 cycles)</td>
</tr>
<tr>
<td>Herculase 5X buffer</td>
<td>10&micro;l</td>
<td>95&deg;C &nbsp; &nbsp;2 min</td>
<td>95&deg;C &nbsp; &nbsp;2 min</td>
</tr>
<tr>
<td>dNTP (10mM)</td>
<td>1.25&micro;l</td>
<td>95&deg;C &nbsp; &nbsp;20 sec</td>
<td>95&deg;C &nbsp; &nbsp;20 sec</td>
</tr>
<tr>
<td>F primer (10&micro;M)</td>
<td>1.25&micro;l</td>
<td>39&deg;C&nbsp; &nbsp; 20 sec</td>
<td>55&deg;C &nbsp; &nbsp;20 sec</td>
</tr>
<tr>
<td>R primer (10&micro;M)</td>
<td>1.25&micro;l</td>
<td>72&deg;C &nbsp; &nbsp;2 min</td>
<td>72&deg;C &nbsp; &nbsp;2 min</td>
</tr>
<tr>
<td>Herculase pol</td>
<td>1&micro;l</td>
<td>72&deg;C &nbsp; &nbsp;3 min</td>
<td>72&deg;C &nbsp; &nbsp;3 min</td>
</tr>
<tr>
<td>H<sub>2</sub>O</td>
<td>To 50&micro;l</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>
<br /><br />* Modify protocol accordingly if using a different polymerase. Significant PCR optimization may be necessary for each sample type. Performing multiple reactions per sample (typically four 50 &micro;l reactions for 16S, four 50 &micro;l reactions for 23S, etc) and pooling the products afterwards may be necessary. High PCR yields (e.g., ~250-500 ng/&micro;l) are needed for the next step (in vitro transcription). <br /><br /><strong>B2</strong>: In vitro transcription (w/ T7 RNA polymerase) of biotin-labeled antisense RNA probes<br />---based on MEGAscript<sup>TM</sup> High Yield Transcription kit (Ambion)<br /><br />For a standard&nbsp;20 &micro;l reaction*:<br /><br />
<table>
<tbody>
<tr>
<td>PCR amplicons (step B1, suggest 250-500 ng)*</td>
<td>1&micro;l</td>
</tr>
<tr>
<td>ATP (75 mM)</td>
<td>2&nbsp;&micro;l</td>
</tr>
<tr>
<td>GTP (75 mM)</td>
<td>2&nbsp;&micro;l</td>
</tr>
<tr>
<td>CTP (75 mM)</td>
<td>1.5&nbsp;&micro;l</td>
</tr>
<tr>
<td>UTP (75 mM)</td>
<td>1.5&nbsp;&micro;l</td>
</tr>
<tr>
<td>Biotin-11-CTP (10 mM, Roche&nbsp;04739205001)</td>
<td>3.75&nbsp;&micro;l</td>
</tr>
<tr>
<td>Biotin-16-UTP (10 mM, Roche 11388908910)</td>
<td>3.75&nbsp;&micro;l</td>
</tr>
<tr>
<td>10X buffer</td>
<td>2&nbsp;&micro;l</td>
</tr>
<tr>
<td>SUPERase&bull;InTM RNase inhibitor</td>
<td>0.5&nbsp;&micro;l</td>
</tr>
<tr>
<td>T7 RNA polymerase</td>
<td>2&nbsp;&micro;l</td>
</tr>
</tbody>
</table>
<br />*Use Speedvac to concentrate PCR amplicons down to 250 ng/&micro;l<br /><br />* The IVT reaction is linear and therefore dependent on starting DNA concentration. A 4-6 hr reaction with 250-500 ng of starting template typically yields high probe concentrations (e.g., 1500-2000 ng/&micro;l). If yield is low, the reaction volume can be increased to 40 &micro;l. Assuming ~50% GC, ~1 in 8 nucleotides should be biotin-labeled in the resulting aRNA probes. <br /><br /><strong>B3</strong>. rRNA subtraction with biotinylated aRNA<br /><br />This step binds biotinylated aRNA probes (step B2) to rRNA in the total RNA sample. The labeled ds rRNA is then removed via hybridization to streptavidin-coated magnetic beads (NEB S1420S), followed by magnetic separation (via a separation rack; e.g., DynaMag<sup>TM</sup>-Spin Magnet, Invitrogen 123-20D).&nbsp; Depending on the rRNA concentration in the sample, the ratio of probe to template RNA may need to optimized. To start, we recommend a probe-to-RNA ratio of 2:1, and a reaction volume of 50 &micro;l in 1X sodium chloride-citrate (SSC) and 20% formamide.&nbsp; For bacterioplankton samples, we&rsquo;ve found that &gt; 20% formamide inhibits probe-bead binding, and &lt; 20% allows non-specific binding (non-target RNA to beads).&nbsp; The procedure obviously involves a significant reduction in RNA concentration. Depending on downstream applications (e.g., RNA amplification), we recommend using as much starting RNA template as possible (ideally 250-500 ng) however we can usually generate enough subtracted RNA for ScriptSeq using as little as 100 ng starting RNA template. <br /><br /><br /><strong>Part C: cDNA synthesis and Illumina library prep</strong><br />---based on EpiCenter ScriptSeq v2 RNA-Seq library preparation kit<br /><br />Notes:&nbsp;<br />- &nbsp;Remove all components except enzymes and Finishing solution, allow to thaw at RT and store on ice. Centrifuge briefly before use. <br />- &nbsp;A thermal cycler with heated lid is recommended for fragmentation and PCR <br />- &nbsp;For cDNA synthesis steps, leave lid of PCR machine open, use Styrofoam lid instead <br />- &nbsp;Otherwise, follow standard ScriptSeq protocol, reproduced below<br /><br /><strong>C1</strong>. Fragment RNA and anneal cDNA synthesis primer<br /><br /><strong>C2</strong>. Synthesize cDNA<br />Note: Use sytrofoam lid on thermocycler, not heated lid for C2-C3<br /><br />Thermocycler setting for part C2:<br /><br />25&deg;C for 5 minutes<br />42&deg;C for 20 minutes<br />37&deg;C Pause<br />37&deg;C for 10 minutes<br />95&deg;C for 3 minutes<br />25&deg;C Pause<br /><br /><strong>C3</strong>. Terminal-tag the cDNA<br />Note: Use sytrofoam lid on thermocycler, not heated lid for C2-C3. Thoroughly mix the terminal-tagging premix before use.<br /><br />Termocycler settings for Part C3:<br /><br />25&deg;C for 15 minutes<br />95&deg;C for 3 minutes<br />4&deg;C hold or ice<br /><br /><strong>C4</strong>. Purify the cDNA<br /><br /><strong>C5</strong>. Amplify the library and add an index<br />Notes:&nbsp;Typically, 12-15 cycles of PCR are performed, more can be done to increase library yields if needed. For 500 pg-5ng input, do 15 PCR cycles. To add an index, replace the Reverse PCR primer included with this kit with one of the ScriptSeq Index PCR primers, available from EpiCenter. <br /><br /><strong>C6</strong>. Purify the RNA-Seq library<br /><br /><strong>At this point, the samples are ready for pooling (check forcompatible indices) and sequencing according to the latest MiSeq sequencing protocol. For v2 sequencing kits we recommend a final pooled library concentration of 6 pM to get a 800K cluster density.</strong>

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