TITLE: yeast single cell RNA-seq (yscRNA-seq)
AUTHORS: Mariona Nadal, Saiful Islam, Wu Wei, Pablo Latorre, Michelle Nguyen, Eulàlia de Nadal, Francesc Posas, Lars M. Steinmetz

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. [Cell growth]
Grow the desired pre-inoculum of your desired yeast strain in their corresponding media O/N.To profile exponentially growing cells, we recommend the initial culture not to grow over OD660=1
?. [Cell growth]
Next morning. Dilute your cells to OD660= 0.05 in the corresponding media and allow for at least 2 cell divisions prior to sorting.
?. [Single cell isolation by FACS sorting]
Prepare 96/384 well plates* containing ** in each to fix cells immediately from sorting* During protocol optimization we recommend using break-away plates  (EK-75118 ). Check with your facility the compatibility of the plates.** this is the minimum volume required for our sorting facility/plates. Check with your facilityNote: We have obtained the same results sorting cells directly into 5 ul of cell capturing solution and lysis (see below). If doing so, prepare plates right before sorting and keep them on
[Absolute Ethanol]
[ice]
?. [Single cell isolation by FACS sorting]
Dilute cells prior to sorting to OD=0.05 in of media and vortex vigurosuly to separate cell clumps.* At this step propidium idodide (PI) can be added to check for cell viability
3 ml
?. [Single cell isolation by FACS sorting]
At the FACS facility, filter cells with Cell Strainer Tubes (check with your facility which tubes they prefer) and put cells in the appropiate sorting tube for live single cell sorting.
?. [Single cell isolation by FACS sorting]
Check the alignment of the plate with the sorter. For example, this can be done by sorting a drop into a covered plate and look for the droplet would fall inside each well.
?. [Single cell isolation by FACS sorting]
Sort live single yeast into the each well of the plates and leave one well (we ususally do H12 as empty/not sorted) asnegativecontrol.* Should you want to do a positive control, sort 100 cells into one well (we usually do A1).
?. [Single cell isolation by FACS sorting]
Cover plates with aluminim seal if plates will not be processed right away or with temporary plastic lid.
?. [Single cell isolation by FACS sorting]
Quickly spin plates to collect cells at the bottom
?. [Single cell isolation by FACS sorting]
Let EtOH avaporate in a sterile environment (stril hood) for
[maximum]
?. [Single cell isolation by FACS sorting]
Once EtOH is completely evaporated, add of cell capturing solution and lysis. Spin down and freeze immediately.Regardless if cells are sorted into EtOH or cell capturing and lysis solution, frozen plates can be stored at -80ºC for at least 6 months. Cell lysis solution:  ABC1  <span style="font-size:9.0pt;font-family:Arial;mso-fareast-font-family:&quot;Times New Roman&quot;;
  color:black">Reagents
    <span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:black">Reference
  Volume (1 rxn)2  <span lang="ES" style="font-size:9.0pt;font-family:Arial;mso-fareast-font-family:&quot;Times New Roman&quot;;
  color:black;mso-ansi-language:ES">1% Triton X-100<span style="font-size:9.0pt;font-family:Arial;mso-fareast-font-family:&quot;Times New Roman&quot;;
  color:black">
    <span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">X100-1L<span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">
  0.053  <span lang="ES" style="font-size:9.0pt;font-family:Arial;mso-fareast-font-family:&quot;Times New Roman&quot;;
  color:black;mso-ansi-language:ES">UMI_Oligo dT_T31(100 <span lang="ES" style="font-size:9.0pt;font-family:Symbol;mso-fareast-font-family:
  &quot;Times New Roman&quot;;mso-bidi-font-family:Arial;color:black;mso-ansi-language:
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  mso-fareast-font-family:&quot;Times New Roman&quot;;color:black;mso-ansi-language:ES">M)
    <span style="font-size:11.0pt;font-family:
  Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;mso-bidi-font-family:&quot;Times New Roman&quot;;
  color:#0563C1">IDT<span style="font-size:11.0pt;font-family:
  Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;mso-bidi-font-family:&quot;Times New Roman&quot;;
  color:#0563C1">
  0.24  <span style="font-size:9.0pt;font-family:Arial;mso-fareast-font-family:&quot;Times New Roman&quot;;
  color:black">dNTP 25 mM
    <span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">R0181<span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">
  0.685  <span style="font-size:9.0pt;font-family:Arial;mso-fareast-font-family:&quot;Times New Roman&quot;;
  color:black">100 mM DTT
    <span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">18064014<span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
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  0.56  <span style="font-size:9.0pt;font-family:Arial;mso-fareast-font-family:&quot;Times New Roman&quot;;
  color:black">Zymolyase 100T (100 mg/ml)
    <span style="font-size:
  11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">37340-57-1<span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">
  0.67  <span lang="ES" style="font-size:9.0pt;font-family:Arial;mso-fareast-font-family:&quot;Times New Roman&quot;;
  color:black;mso-ansi-language:ES">RNase Inhibitor (40 U/ml)<span style="font-size:9.0pt;font-family:Arial;mso-fareast-font-family:&quot;Times New Roman&quot;;
  color:black">
    <span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">&nbsp;2313A<span style="font-size:
  11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">
  0.128<span style="font-size:9.0pt;font-family:Arial;mso-fareast-font-family:&quot;Times New Roman&quot;;
  color:black">RNase-Nuclease free H2O<span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">10977035<span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">2.7699<span style="font-size:9.0pt;font-family:Arial;mso-fareast-font-family:&quot;Times New Roman&quot;;
  color:black">ERCC (1:1000,0000) (5000 molecules)  <span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">4456740<span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">
  0.08110Total5
[yeast cell lysis solutuin]
?. [Lysis reaction]
Do the follwoing lysis cycle from fresh sorted or frozen plates and quickly proceed to add RT reaction for 1st strand cDNA synthesisIncubate at 30ºC for at least 10 minutes *3 minutes at 72ºC1 minutes at 4C* We have changed the length of cell lysis up to 30 minutes
?. [1st strand cDNA synthesis]
5 Add   ABC1ReagentsVolume (1 rxn)Reference25X SuperScript Stand Buffer2  <span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">18064014<span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">
  3MgCl2 (1M)0.06  <span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">AM9530G<span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">
  4Betaine (5M)1.6  <span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">61962<span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">
  5UMI_TSO 100uM0.2  <span style="font-size:11.0pt;font-family:
  Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;mso-bidi-font-family:&quot;Times New Roman&quot;;
  color:#0563C1">IDT<span style="font-size:11.0pt;font-family:
  Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;mso-bidi-font-family:&quot;Times New Roman&quot;;
  color:#0563C1">
  6200 U/ul SuperScript II0.25  <span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">18064014<span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">
  7RNase inhibitor (40U/ml)0.125  <span style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">&nbsp;2313A<span style="font-size:
  11.0pt;font-family:Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;
  mso-bidi-font-family:&quot;Times New Roman&quot;;color:#0563C1">
  8RNase-Nuclease free H2O0.765109770359Total5
[Reverse transcription mix (RT mix)]
?. [1st strand cDNA synthesis]
Spin down the plate and perform the following cycles42ºC for 90 mins70ºC for 15 mins4ºC forever
?. [Library amplification]
Add  for library amplification  AB1ReagentsVolume (1 rxn)210X Advantage 2 PCR buffer2.53dNTPs (25mm)0.44UMI_PCR (10uM)1.2550X Advantage 2 polymerase mix0.56H2010.47Total15
[PCR mix]
?. [Library amplification]
Spin down the plate and do the following cycleshold at 4ºC overnight (if necessary). This is usually a safe stopping point
?. [qPCR validation ]
Perform a 1:20 dilution of the amplified library to check the percentage of positive libraries. Use your favorite protocol, and use as a target primers to a housekeeping gene or ERCCs.This step is specially useful during the protocol set up as it allows to isnpect the efficiency of the protocol before moving forward.  AB1ReagentVolume (μl)2Sybergreen 2X2.53Primer mix*0.1254H2O1.3755DNA1 * Primer mix is a mmix of Fw and Rv primer (10µM each)1 cycle: 95ºC 10 min40 cycles: 95ºC 30 sec, 58ºC 30 sec and 72ºC 30 sec1 cycle: 95ºC 15 sec, 60ºC 15 sec and 95ºC 15 sec (melting curve)Primer sequence for qPCR:SOMN17 Fw_TDH3_probe TCGTCAAGTTGGTCTCCTGGSOMN18 Rv_TDH3_probe GGCAACGTGTTCAACCAAGT  SOMN21 Fw_ADH1_probe TGGTGCCAAGTGTTGTTCTGSOMN22 Rv_ADH1_probe GGCGAAGAAGTCCAAAGCTTSOMN310 Fw_5_ERCC_00130 CGGAAAAGTACTGACCAGCGSOMN311 Rv_5_ERCC_00130 TGCCAATGACTTCAGCTGACA good plate will have around 70% positive wells, as far as it’s around 50% it should be OK. Rearrange positive cells into a new plate to proceed to tagmentation.
?. [dscDNA library clean up]
Before commencing the purification steps, equilibrate Ampure XP beads at room temperature for 15 min, and then vortex well for several seconds. Add  (1:0.6 ratio) to each sample (each tube might have slightly different volumes due to evaporation).*Do not increase the volume of beads in the purification step above the 1:1 ratio. A less-than-standard amount of beads ensures that primer dimer carryover is kept to a minimum.
[Ampure XP beads]
?. [dscDNA library clean up]
Mix by pipetting up and down ten times or until the solution appears homogeneous. Transfer solutions to a 96-well plate with compatible magnet stand
?. [dscDNA library clean up]
Incubate the mixture for 10 min at room temperature to let the DNA bind to the beads.
?. [dscDNA library clean up]
Place the 96-well plate on the magnetic stand for 5 min or until the solution is clear and the beads have been collected
?. [dscDNA library clean up]
While samples are on the magnet, carefully remove the liquid without disturbing the beads.
?. [dscDNA library clean up]
Wash the beads with 200 l of 80% (vol/vol) ethanol solution. Incubate the samples for 30 sec and then remove the ethanol.  *It is important that the ethanol solution is freshly prepared every time, as ethanol absorbs moisture from the environment, thus changing the final concentration. Repeat ethanol washing.
?. [dscDNA library clean up]
Repeat ethanol washing one more time.
?. [dscDNA library clean up]
Remove any trace of ethanol and let the beads dry completely, leaving the plate at room temperature for 5 min or until a small crack appears on the surface of the beads.*Avoid over drying the beads because this will make their resuspension in the designated buffer more difficult. ** As a precaution, cover the plate during this step or protect it from any possible source of contamination or air flows that might disperse the beads around the well, thus leading to cross-contamination between adjacent wells especially.
?. [dscDNA library clean up]
When beads are dried, elute dscDNA libraries with  (19086)
[elution buffer (EB buffer Qiagen)]
?. [dscDNA library clean up]
Remove plate from the magnet and mix vigurously by pipetting up and down x3 times to resuspend beads out of the magnet
?. [dscDNA library clean up]
Place the plate on the magnetic stand and leave it for 2 min or until the solution appears clear and beads have accumulated in a corner of the well.
?. [dscDNA library clean up]
Recover 15 ml of SPN from each well and transfer to a new plate. Label correctly as this plate will be stored.SAFE STOPPING POINT: cDNA llibraries can be stored at -20ºC before proceeding to tagmentation.
?. [dscDNA library/concentration size validation]
Run of several purified dscDNA libraries to check the size distribution and estimate of concentrations using a High Sensitivity DNA ChIP (2100 Bioanalyzer). qPCR validation using a housekeeping gene is valuable to guide well selection. Always run your negative control.
1 µl
?. [Adaptor annealing for tagmentation]
In order to load Tn5 with cell-specific adaptors, these need to be annealed as dsDNA cell-specific adaptors.To anneal the adapters mix in a 96 well plate:Mix UMI-TN5-U  (100 µM) and UMI-TN5_1 (µM) to 96 in TE 1X to final concentration 50 µM (each), a 1:1 dilution. Primer annealing thermocycler: 95ºC for 3 minutes and gradually cool down to room temperature (0,5 ºC/sec). This plate can be stored at -20ºC for several months and used to lead several rounds of Tn5.
?. [Tn5 loading with cell-specific adaptors]
Prepare the following mix and aliquot ,Prepare the following mix and aliquot of the mix (except for adapters) into a new 96 well plate. Then add of each adaptor to each well.  ABC1  <b style="mso-bidi-font-weight:
  normal"><span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:
  major-latin;mso-hansi-theme-font:major-latin">Reagent<span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:major-latin;
  mso-hansi-theme-font:major-latin">
    <span style="font-size:9.0pt;
  font-family:Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:
  major-latin">Volume <span style="font-size:9.0pt;font-family:Calibri;
  mso-ascii-theme-font:major-latin;mso-hansi-theme-font:major-latin">
  Observations2  <span style="font-size:
  9.0pt;font-family:Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:
  major-latin">50 µM adapter (96 different)
    <span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:
  major-latin;mso-hansi-theme-font:major-latin">1.25 µL
  add this later individually to each well3  <span style="font-size:
  9.0pt;font-family:Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:
  major-latin">80% Glycerol
    <span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:
  major-latin;mso-hansi-theme-font:major-latin">6.25 µL
  4  <span style="font-size:
  9.0pt;font-family:Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:
  major-latin">50 µM Tn5 transposase
    <span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:
  major-latin;mso-hansi-theme-font:major-latin">1.25 µL
  Hennig, Bianca P., et al. "Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol." G3: Genes, Genomes, Genetics (2017): g3-300257.5  <span style="font-size:
  9.0pt;font-family:Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:
  major-latin">Nuclease-free water
    <span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:
  major-latin;mso-hansi-theme-font:major-latin">1.25 µL
  6  <b style="mso-bidi-font-weight:
  normal"><span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:
  major-latin;mso-hansi-theme-font:major-latin">Total volume<span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:major-latin;
  mso-hansi-theme-font:major-latin">
    <span style="font-size:9.0pt;
  font-family:Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:
  major-latin">10 µL<span style="font-size:9.0pt;font-family:Calibri;
  mso-ascii-theme-font:major-latin;mso-hansi-theme-font:major-latin">
8 µl
1 µl
?. [Tn5 loading with cell-specific adaptors]
Incubate 37°C for one hour and freeze  if not going to be used right away.* Note, this Tn5-loaded plate can be safely stored for a 1-2 weeks at . However leaving the loaded plate on  ice  will significantly reduce Tn5 activity and will result in inefficient tagmentation.
?. [Tagmentation]
Prepare the following mix  AB1  <b style="mso-bidi-font-weight:
  normal"><span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:
  major-latin;mso-hansi-theme-font:major-latin">Reagent<span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:major-latin;
  mso-hansi-theme-font:major-latin">
    <span style="font-size:9.0pt;
  font-family:Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:
  major-latin">Volume (per well)<span style="font-size:9.0pt;
  font-family:Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:
  major-latin">
  2  <span style="font-size:
  9.0pt;font-family:Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:
  major-latin">Harvested DNA
    <span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:
  major-latin;mso-hansi-theme-font:major-latin">6 µL
  3  <span style="font-size:
  9.0pt;font-family:Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:
  major-latin">Nuclease-free water
    <span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:
  major-latin;mso-hansi-theme-font:major-latin">8 µL
  4  <span style="font-size:
  9.0pt;font-family:Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:
  major-latin">2x TAPS buffer
    <span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:
  major-latin;mso-hansi-theme-font:major-latin">2 µL
  5  <span style="font-size:
  9.0pt;font-family:Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:
  major-latin">100% DMF
    <span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:
  major-latin;mso-hansi-theme-font:major-latin">2 µL
  6  <span style="font-size:
  9.0pt;font-family:Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:
  major-latin">10x Transposome 
    <span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:
  major-latin;mso-hansi-theme-font:major-latin">2.0 µL
  7  <b style="mso-bidi-font-weight:
  normal"><span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:
  major-latin;mso-hansi-theme-font:major-latin">Total volume<span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:major-latin;
  mso-hansi-theme-font:major-latin">
    <span style="font-size:9.0pt;
  font-family:Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:
  major-latin">20 µL<span style="font-size:9.0pt;font-family:Calibri;
  mso-ascii-theme-font:major-latin;mso-hansi-theme-font:major-latin">
?. [Tagmentation]
Incubate for 55ºC for 5 min -> 3 min 85ºC to inactivate Tn5 and then cool to * The tagmentation time can vary depending on the Tn5 purification batch.
?. [5' capture through Streaptavidin beads]
Do a 1:20 dilution of MyOne Streptavidin for the total number of samples (.(Example:  for 20 samples)2XBWT Buffer:10 mM Tris-HCl pH7.51mM EDTA2M NaCl 0.02% Tween-20
[of beads/sample)]
[beads]
?. [5' capture through Streaptavidin beads]
Wash MyOne  beads x2 with 2XBWT buffer and resupsend with  20X more volume than the original volume of beads with 2XBWT(Example:  for 20 samples, will be finally resuspended with
[beads]
[2XBWT]
?. [5' capture through Streaptavidin beads]
Add 20 µL beads to each well and incubate at RT for 5 min at room temprature
?. [5' capture through Streaptavidin beads]
Pool all samples into a single collecting tube (1.5 or 2 ml)
?. [5' capture through Streaptavidin beads]
Place collecting tube in magnetic rack and allow time enough for the solution to be completely clear
?. [5' capture through Streaptavidin beads]
Wash beads once with TNT buffer TNT Buffer:20 mM Tris pH 7.5 50 mM NaCl,0.02% Tween
100 µl
?. [5' capture through Streaptavidin beads]
Wash the beads once in  Qiaquick PB  discard SPN
100 µl
?. [5' capture through Streaptavidin beads]
Wash beads 3x with TNT buffer again, discard SPN
100 µl
?. [Removing 3' end fragments]
Resuspend the beads in the following mix:   AB1  <b style="mso-bidi-font-weight:
  normal"><span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:
  major-latin;mso-hansi-theme-font:major-latin">Reagent<span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:major-latin;
  mso-hansi-theme-font:major-latin">
    <span style="font-size:9.0pt;
  font-family:Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:
  major-latin">Volume<span style="font-size:9.0pt;font-family:Calibri;
  mso-ascii-theme-font:major-latin;mso-hansi-theme-font:major-latin">
  2  <span style="font-size:
  9.0pt;font-family:Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:
  major-latin">CutSmart buffer
    <span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:
  major-latin;mso-hansi-theme-font:major-latin">10 µL
  3  <span style="font-size:
  9.0pt;font-family:Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:
  major-latin">PvuI-HF enzyme (20 U/µL)
    <span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:
  major-latin;mso-hansi-theme-font:major-latin">2 µL
  4  <span style="font-size:
  9.0pt;font-family:Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:
  major-latin">Nuclease-free water
    <span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:
  major-latin;mso-hansi-theme-font:major-latin">88 µL
  5  <b style="mso-bidi-font-weight:
  normal"><span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:
  major-latin;mso-hansi-theme-font:major-latin">Total volume<span style="font-size:9.0pt;font-family:Calibri;mso-ascii-theme-font:major-latin;
  mso-hansi-theme-font:major-latin">
    <span style="font-size:9.0pt;
  font-family:Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:
  major-latin">100 µL<span style="font-size:9.0pt;font-family:Calibri;
  mso-ascii-theme-font:major-latin;mso-hansi-theme-font:major-latin">
?. [Removing 3' end fragments]
Incubate at 37°C for one hour with interval mix during the incubation (to avoid beads precipitation); 2 min without mix, 30 sec 1000 rpm mix.
?. [Removing 3' end fragments]
Wash the beads three times in TNT.
100 µl
?. [Eluting single stranded cDNA library]
Resuspend in 30 µL Nuclease-free water.
30 µl
?. [Eluting single stranded cDNA library]
Incubate 10 min at 70°C, 850 rpm mix.
?. [Eluting single stranded cDNA library]
Bind the beads to the magnet immediately and collect the supernatant.
?. [sscDNA cleanup]
Add 54µl of room temperature Ampure XP beads to 30µ sscDNA library
[Ampure XP beads]
?. [sscDNA cleanup]
Incubate 10 min at RT.
?. [sscDNA cleanup]
Bind the beads to the magnet for 1min and discard supernatant or until solution is completely clear.
?. [sscDNA cleanup]
Wash once with 200 µL fresh 80% ethanol for 20-30 sec. Let the beads be bound to the magnet the entire time.
?. [sscDNA cleanup]
Dry the beads for up to 2 min.
?. [sscDNA cleanup]
Resuspend in 30 µL Qiagen EB buffer and incubate 5 min at RT.
[EB]
?. [sscDNA cleanup]
Bind the beads 1 min and transfer SPN to a new tube.
?. [Library Concentration]
To quantify library concentration, set up a KAPA quantification reaction with a 1:100 and 1:1000 dilutions of the eluted cDNA library.  AB1ReagentVolume (1 rxn)2KAPA SYBR® FAST qPCR Master Mix containing Primer Premix 12 µl3PCR-grade water 4 µ4Diluted library DNA or DNA Standard4 µl5TOTAL20 µlThis kit can be subsituted by your favorite quantification method or by a qPCR using  P5-P7 primer pairs with known standards (PhiX is strongly recommended) sybergreen 2X mastermix.
?. [Library Concentration]
Use the qPCR to calculate library quantification using the template provided by KAPA biosystems or the instructions provided from your manufacturer.We have used KAPA, NEB and homemade systems with similar results.
?. [Library Size]
Sett up a separate PCR to run a bioanalyzer to determine the final size distribution. Prepare the following mix:  AB1  KAPA SYBR Q-PCR Mastermix ABI
  Prism
    10 µL
  2  Primers mix (10 X)
    2 <span style="font-size:9.0pt;
  font-family:Symbol;mso-ascii-font-family:Cambria;mso-ascii-theme-font:minor-latin;
  mso-hansi-font-family:Cambria;mso-hansi-theme-font:minor-latin;mso-char-type:
  symbol;mso-symbol-font-family:Symbol"><span style="mso-char-type:symbol;
  mso-symbol-font-family:Symbol">ml
  3  <span style="font-size:
  9.0pt">Water
    6 µL
  4  <span style="font-size:
  9.0pt">DNA without dilution
    2 µl
  5  <b style="mso-bidi-font-weight:
  normal">Total volume
    20
  µl
?. [Library Size]
Run the same PCR as in step 58 but for 11 cycles
?. [Library Size]
Run 1 µL into a High sensitivity DNA CHIP to obtain an average library size based on the Bioanalyzer profile.
?. [Library Concentration]
qPCR cycling conditions for KAPA and homemade Sybergreen  ABC1TemperatureTime295ºC5 min395ºC30 secrepeat this for 30 cycles for qPCR quantification460ºC45 sec54ºChold
?. [Sequencing]
Sequence the library on the HiSeq 2000 High output using C1-P1-PCR-2 as the Read 1 primer and UMI-TN5-U as the Index read primer.
?. [Sequencing]
To run the libraries on the HiSeq rapid run, us LNA primers. Spike in at primer at 0.5 uM.Index 1 primer into HP8 (position 17) in the HiSeqRead 1 primer into HP9 (position 16) in the Hiseq(double check this information with your sequencing kit/instrument)UMI_PCR_read1: +GAATGA+TACGGCG+ACCA +CCGA+T - custom 250 nmole. DNA oligo, HPLC Purification Index1: CTGT+CT+CTT+ATA+CA +CA+TCTGA+CG+C - custom 250 nmole DNA oligo, HPLC Purification*Note for High Output run custom primers are needed as well but without LNA
?. [Sequencing]
Load around 8-14 pmol of each library per lane. Libraries are single stranded DNA, so no denaturing is required.