TITLE: prime-seq
AUTHORS: Aleksandar Janjic, Lucas Esteban Wange, Johannes Bagnoli, Johanna Geuder, Phong Nguyen, Daniel Richter, Christoph Ziegenhain, Wolfgang Enard

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>prime-seq is a simple and open RNA-seq method, which can be easily established in most research labs or facilities. Based on two of the most sensitive single cell RNA-seq methods available, namely smart-seq2 and mcSCRB-seq </span><a href="https://paperpile.com/c/lZdm6I/9O4L+GC4P" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">(Picelli et al. 2013; Bagnoli et al. 2018)</span></a><span>, prime-seq is an adaptable, affordable, robust, and high-throughput option. Additionally, prime-seq can be used on a wide range of model organisms, from apes to zebrafish, and many in between. </span></div></div>

[STEPS]
?. [Sample Collection]
Add cells or tissue to wells
CellsMinimum: 100 cells, Optimum: 10,000 cells Make sure that the same number of cells are used for each sample. Large differences between cells will impact distribution of sequencing reads and can potentially affect normalization.
TissueIf samples are difficult to lyse they should be homogenized using a tissue homogenizer. Tissue should be a relatively small and not exceed more than 1000 ng of RNA. Tissue samples should be normalized by weight and be the same type of tissue. Large differences between tissue samples will impact distribution of sequencing reads and can potentially affect normalization. If you are unsure if the samples will contain the same amount of RNA, it is best to switch to the "Lysate (variable)" case in Step 13.
?. [Sample Collection]
Add of Lysis Buffer to each well of a semi-skirted 96-well PCR plate
100 µl
?. [Sample Collection]
Prepare Lysis Buffer according to the number of samples.  ABC1ReagentWellPlate2RLT Plus Buffer99 µL10.89 mL3β-mercaptoethanol1 µL110 µL4Total
100 µL11 mL
ABC1ReagentWellPlate2RLT Plus Buffer99 µL10.89 mL3β-mercaptoethanol1 µL110 µL4Total
100 µL11 mL
If sample volume exceeds 25 % of total lysate, use 2x TCL buffer 
(Qiagen, #1070498) + 1 % β-mercaptoethanol
?. [Bead Clean Up]
Mix each bulk sample (50 µL per well) with  of Cleanup Beads (22% PEG)
100 µl
?. [Bead Clean Up]
Incubate for  at
[(Room Temp)]
While binding, prepare DNase I Mix (Step 28)
?. [Bead Clean Up]
Place on magnet stand until clear (~3 min) and then discard supernatant
?. [Bead Clean Up]
Wash with  of 80% EtOH while the plate is on the magnet. Discard the supernatant
100 µl
?. [Bead Clean Up]
Repeat wash step once more
?. [Bead Clean Up]
Air dry beads for
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
?. [Sample Collection]
Transfer  of lysate to a new plate, return one plate immediately to -80 C freezer to save as a backup
50 µl
Conversely, one can prepare two plates during sorting with 50 µL of lysis buffer.
?. [DNAse I Digest]
Resuspend beads in  of DNase I Mix
15 µl
?. [DNAse I Digest]
Incubate DNase I Mix and beads for  at
[(Room Temp)]
?. [DNAse I Digest]
Heat inactivate the DNase I by adding  of EDTA (100 mM) and incubating for  at
1 µl
65 °C
?. [DNAse I Digest]
Place plate on magnet stand until clear (~3 min) and discard the supernatant.
?. [DNAse I Digest]
Wash with  of 80% EtOH while the plate is on the magnet. Discard the supernatant
100 µl
?. [DNAse I Digest]
Repeat wash step once more
?. [DNAse I Digest]
Air dry beads for
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
While drying, prepare Reverse Transcription Mix (Step 36).
?. [Reverse Transcription]
Prepare Reverse Transcription Mix  ABC1ReagentWellPlate2Maxima H Minus RT0.15 µL16.5 µL3Maxima RT Buffer (5x)2 µL220 µL4dNTPs (25 mM)0.4 µL44 µL5TSO (E5V7NEXT) (100 uM)0.1 µL11 µL6UltraPure Water2.35 µL258.5 µL7Total5 µL550 µL
ABC1ReagentWellPlate2Maxima H Minus RT0.15 µL16.5 µL3Maxima RT Buffer (5x)2 µL220 µL4dNTPs (25 mM)0.4 µL44 µL5TSO (E5V7NEXT) (100 uM)0.1 µL11 µL6UltraPure Water2.35 µL258.5 µL7Total5 µL550 µL
?. [Reverse Transcription]
Add  Reverse Transcription Mix
5 µl
?. [Reverse Transcription]
Add  of Barcoded oligodT (E3V7NEXT) (10 µM) per well
1 µl
?. [Reverse Transcription]
Incubate for  at
42 °C
?. [cDNA Pooling & Purification]
Pool all wells of one plate into a 2 mL tube
?. [cDNA Pooling & Purification]
Add  of Cleanup Beads (22% PEG) for each sample for a 1:1 ratio (e.g. 240 µL for 24 samples)
10 µl
The EDTA in the Cleanup Beads (22% PEG) will inactivate the RT and make pooling easier due to the color.
?. [cDNA Pooling & Purification]
Incubate for  at  to allow binding of the cDNA onto beads
20 Room temperature
?. [cDNA Pooling & Purification]
Place the tube on the magnet stand until clear (~3 min) and discard supernatant
?. [cDNA Pooling & Purification]
Wash with  of 80% EtOH while the tube is on the magnet. Discard the supernatant
1 mL
Volume of EtOH should be adjusted depending on the number of samples. More samples will require more EtOH to cover the beads completely.
?. [cDNA Pooling & Purification]
Repeat wash step once more
?. [cDNA Pooling & Purification]
Air dry beads for
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
?. [cDNA Pooling & Purification]
Elute the beads in 17 µL of UltraPure Water
?. [Exonuclease I Treatment]
Add  of ExoI Buffer (10x) and  of Exonuclease I. Incubate as follows:  ABC1StepTemperatureTime2Incubation37 C20 min3Heat Inactivation80 C10 min4Storage4 C∞
2 µl
1 µl
ABC1StepTemperatureTime2Incubation37 C20 min3Heat Inactivation80 C10 min4Storage4 C∞
?. [Full length cDNA Amplification]
Prepare Pre Amplification Mix  AB1Reagent1x2KAPA HiFi 2x RM25 µL3Pre-amp Primer (SINGV6) (10 uM)3 µL4UltraPure Water2 µL5Total30 µL
AB1Reagent1x2KAPA HiFi 2x RM25 µL3Pre-amp Primer (SINGV6) (10 uM)3 µL4UltraPure Water2 µL5Total30 µL
?. [Full length cDNA Amplification]
Add  Pre Amplification Mix to sample
30 µl
?. [Full length cDNA Amplification]
Incubate the Pre Amplification PCR as follows:  ABCD1StepTemperatureTimeCycles2Initial Denaturation98 C3 min1 cycle3Denaturation98 C15 sec10 cycles*4Annealing65 C30 sec5Elongation72 C4 min6Final Elongation72 C10 min1 cycle7Storage4 C∞
ABCD1StepTemperatureTimeCycles2Initial Denaturation98 C3 min1 cycle3Denaturation98 C15 sec10 cycles*4Annealing65 C30 sec5Elongation72 C4 min6Final Elongation72 C10 min1 cycle7Storage4 C∞
Adjust the number of cycles based on input (sample number, cell number, or concentration). As a rule of thumb we assume big cells like embryonic stem cells to contain 10 pg of total RNA and small cells like T-cells ~ 1-2 pgAs a general guide we recommend:  AB1Total RNA InputCycles210 ng16350 ng144100 ng125500 ng1061000 ng9
?. [cDNA Bead Purification ]
Mix sample with  Clean Up Beads (22% PEG) for a ratio of 1:0.8
40 µl
?. [cDNA Bead Purification ]
Incubate for  at
[(Room Temp)]
?. [cDNA Bead Purification ]
Place the tube on the magnet stand until clear (~3 min) and discard supernatant
?. [cDNA Bead Purification ]
Wash with  of 80% EtOH while the tube is on the magnet. Discard the supernatant
100 µl
?. [cDNA Bead Purification ]
Repeat wash step once more
?. [cDNA Bead Purification ]
Air dry beads for
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
?. [cDNA Bead Purification ]
Elute cDNA in  UltraPure Water
10 µl
?. [cDNA Quantification and Quality Check]
Quantify the cDNA using the Quant-iT PicoGreen dsDNA assay kit or equivalent Qubit following the manufacturer's protocol. Use 1 μl of clean cDNA for quantification.
?. [cDNA Quantification and Quality Check]
Quality check the cDNA using the Agilent 2100 Bioanalyzer with High Sensitivity DNA Analysis Kits.
Passing the cDNA quality check does not guarantee that the data will be of high quality, however,  if the cDNA fails the quality check it will usually not yield good libraries and will therefore generate lower quality data.
?. [Library Preparation]
Prepare Fragmentation Mix  AB1Reagent1x2Ultra II FS Reaction Buffer1.4 µL3Ultra II FS Enzyme Mix0.4 µL4cDNA (4-8 ng/µL)2.5 µL5TE1.7 µL6Total6 µL
AB1Reagent1x2Ultra II FS Reaction Buffer1.4 µL3Ultra II FS Enzyme Mix0.4 µL4cDNA (4-8 ng/µL)2.5 µL5TE1.7 µL6Total6 µL
Ensure that the Ultra II FS Reaction Buffer is completely thawed. If a precipitate is seen in the buffer, pipette up and down several times to break it up, and quickly vortex to mix. Place on ice until use.
Vortex the Ultra II FS Enzyme Mix for 5-8 seconds prior to use for optimal performance.
?. [Adapter Ligation]
Prepare Adapter Ligation Mix  AB1Reagent1x2NEBNext Ultra II Ligation Master Mix6 µL3NEBNext Ligation Enhancer0.2 µL4prime-seq Adapter (1.5 µM)0.5 µL5Total6.7 µL
AB1Reagent1x2NEBNext Ultra II Ligation Master Mix6 µL3NEBNext Ligation Enhancer0.2 µL4prime-seq Adapter (1.5 µM)0.5 µL5Total6.7 µL
?. [Library Double Size Selection]
Mix Index PCR with  SPRI select beads (ratio of 1:0.52)
26 µl
We use SPRI Select Beads here instead of our home made 22% Clean Up beads for their guaranteed QCed size selection properties.
?. [Library Double Size Selection]
Incubate for  at
[(Room Temp)]
?. [Library Double Size Selection]
Place the plate on the magnet stand until clear and transfer  supernatant to clean well.
76 µl
Be careful not to discard! This is your library.
?. [Library Double Size Selection]
Wash with  of 80% EtOH while the plate is on the magnet. Discard the supernatant
150 µl
?. [Library Double Size Selection]
Repeat wash step once more
?. [Library Double Size Selection]
Air dry beads for
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
?. [Library Double Size Selection]
Elute in  UltraPure Water.
15 µl
?. [Library QC]
Quantify and quality control the library using the Agilent 2100 Bioanalyzer with High Sensitivity DNA Analysis Kits.
Bulk libraries often yield high concentrations, which should be diluted to get accurate molarity measurements on the Bioanalyzer. Ideally, do not load more than 2 ng onto the chip.
Successful libraries will typically be 2-5 ng/μl concentration
?. [Proteinase K Digest]
Add  Proteinase K (20 mg/mL) and  EDTA (25 mM) to each well
1 µl
1 µl
?. [Proteinase K Digest]
Incubate for  at  and then heat inactivate the Proteinase K for  at
50 °C
75 °C
?. [DNAse I Digest]
Prepare DNase I Mix   ABC1ReagentWellPlate2DNase I1 µL110 µL3DNase I Buffer (10x)2 µL220 µL4Bead Binding Buffer (2x)10 µL1.1 mL5H2O2 µL220 µL6Total15 µL1.65 mL
ABC1ReagentWellPlate2DNase I1 µL110 µL3DNase I Buffer (10x)2 µL220 µL4Bead Binding Buffer (2x)10 µL1.1 mL5H2O2 µL220 µL6Total15 µL1.65 mL
?. [Library Double Size Selection]
Incubate for  and then place on magnet until clear. Transfer eluted library to new well.
Stopping point. The libraries can be safely stored at   until they wil be QCed and sequenced.
?. [Sequencing]
Samples should be submitted according to your Sequencing Facility specifications. At least 8 cycles are required for the Index Read (i7) and 28 cycles for the Read 1 (BC+UMI). Read 2 (DNA) should be adjusted based on the quality of the genome being mapped to, but for human and mouse 50 cycles is sufficient.Some potential sequencing options:  ABCDEF1ReadRead 1Read 2Index Read (i7)Index Read (i5)Kit2HiSeq2811480HiSeq 3000/4000 150 cycles3NextSeq285680NextSeq 500/550 HiOut v3 75 cycle4NovaSeq289488NovaSeq SP v1.5 100 cycle
ABCDEF1ReadRead 1Read 2Index Read (i7)Index Read (i5)Kit2HiSeq2811480HiSeq 3000/4000 150 cycles3NextSeq285680NextSeq 500/550 HiOut v3 75 cycle4NovaSeq289488NovaSeq SP v1.5 100 cycle
?. [Library Preparation]
Vortex the Fragmentation Mix for and immediately proceed to step 70
?. [Library Preparation]
Incubate the Fragmentation reaction as follows:  ABC1StepTemperatureTime2Pre-Cool4 C∞3Fragmentation37 C5 min4A Tailing and Phosphorylation65 C30 min5Storage4 C∞
ABC1StepTemperatureTime2Pre-Cool4 C∞3Fragmentation37 C5 min4A Tailing and Phosphorylation65 C30 min5Storage4 C∞
Set heated lid to 75° C. Make sure the lid is at the correct temperature before you start the reaction.Skip the first incubation step once you have added your samples.
?. [Adapter Ligation]
Add   Adapter Ligation Mix to each replicate
6.7 µl
?. [Adapter Ligation]
Incubate for  at
20 °C
Turn off heated lid
?. [Adapter Ligation]
Elute samples in  0.1X TE (dilute 1X TE Buffer 1:10 in water) for
10.5 µl
?. [Library PCR]
Transfer samples to clean wells
?. [Library PCR]
Add  of Index Primer (i7, 5 uM) to each well
1 µl
This is the unique index that will be used for demultiplexing libraries.
?. [Library PCR]
Add  of Library PCR Mix to each well
13.5 µl
?. [Library PCR]
Prepare Library PCR Mix  ABC1Reagent1x5x2NEBNext Ultra II Q5 Master Mix12.5 µL62.5 µL3P5NEXTPT5 primer (5uM stock)1 µL5 µL4Total13.5 µL67.5 µL
ABC1Reagent1x5x2NEBNext Ultra II Q5 Master Mix12.5 µL62.5 µL3P5NEXTPT5 primer (5uM stock)1 µL5 µL4Total13.5 µL67.5 µL
Although scaled down, there will not be sufficient Q5 Master Mix (M0544L) in the kit. This item will have to be ordered separately.
Instead of the universal P5NEXTPT5 primer, one can use unique i5 Indices for each sample. This is recommended if using a sequencer with patterned flow cells to account for index hopping.
?. [Library PCR]
Incubate the Library PCR reaction as follows:  ABCD1StepTemperatureTimeCycles2Initial Denaturation98 C30 sec1 cycle3Denaturation98 C10 sec10 cycles*4Annealing/Elongation65 C1 min 15 sec5Final Elongation65 C5 min1 cycle6Storage4 C∞
ABCD1StepTemperatureTimeCycles2Initial Denaturation98 C30 sec1 cycle3Denaturation98 C10 sec10 cycles*4Annealing/Elongation65 C1 min 15 sec5Final Elongation65 C5 min1 cycle6Storage4 C∞
Adjust the number of cycles based on total cDNA input.As a general guide we recommend:  AB1cDNA InputCycles220 ng10310 ng1145 ng12
?. [Library Double Size Selection]
Mix supernatant with  SPRI select beads (ratio of 1:0.72)
10 µl
?. [Library Double Size Selection]
Incubate for  at
[(Room Temp)]
?. [Library Double Size Selection]
Place the plate on the magnet stand until clear and discard supernatant.
?. [Prepare Cleanup Beads (22% PEG)]
Prepare PEG Solution (22%) by adding all ingredients to a 50 mL falcon tube  AB1&nbsp;ReagentAmount2PEG 800011 g
3NaCl (5M)10 mL4Tris-HCl (1M, pH 8.0)500 μL5EDTA (0.5M)100 μL
6IGEPAL (10% solution)50&nbsp;μL7Sodium Azide (10% solution)250&nbsp;μL8UltraPure Waterup to&nbsp;49 mL9Total&nbsp;49&nbsp;mL
AB1&nbsp;ReagentAmount2PEG 800011 g
3NaCl (5M)10 mL4Tris-HCl (1M, pH 8.0)500 μL5EDTA (0.5M)100 μL
6IGEPAL (10% solution)50&nbsp;μL7Sodium Azide (10% solution)250&nbsp;μL8UltraPure Waterup to&nbsp;49 mL9Total&nbsp;49&nbsp;mL
Do not add the total amount of water until after PEG is completely solubilized
?. [Prepare Cleanup Beads (22% PEG)]
Incubate at  and vortex regularly until PEG is completely dissolved
40 °C
?. [Prepare Cleanup Beads (22% PEG)]
Resuspend Sera-Mag Speed Beads carefully and pipette  of bead suspension into a 1.5 mL tube
1000 µl
?. [Prepare Cleanup Beads (22% PEG)]
Place on magnet stand and remove storage buffer
?. [Prepare Cleanup Beads (22% PEG)]
Add  of TE Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and resuspend beads
1000 µl
?. [Prepare Cleanup Beads (22% PEG)]
Place on magnet stand and remove supernatant
?. [Prepare Cleanup Beads (22% PEG)]
Repeat wash step one more time
?. [Prepare Cleanup Beads (22% PEG)]
Add  TE Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and resuspend beads
900 µl
?. [Prepare Cleanup Beads (22% PEG)]
Add the washed Sera-Mag Speed Beads to the PEG Solution (22%) and mix well
The final Cleanup Beads (22% PEG) can be aliquoted and stored at   for up to six months
?. [Lysate vs Extracted RNA]
prime-seq can be used on lysate or extracted RNA. It is essential, however, that the samples either have the same input or that they are normalized after the RNA is extracted, otherwise sequencing depth per sample will be impacted. Based on your starting material, please follow one of the following cases:
?. [Preparation]
Clean all surfaces and pipettes with RNase Away.
?. [Prepare Bead Binding Buffer]
Prepare Bead Binding Buffer (2x)  AB1Reagent2PEG 80001.1 g3NaCl (5 M)1 mL4Tris-HCl (1 M, pH 8.0)50 µL5Igepal (10% solution)5 µL6Sodium Azide (10% solution)25 µL7H2Oto 5 mL8Total5 ml
AB1Reagent2PEG 80001.1 g3NaCl (5 M)1 mL4Tris-HCl (1 M, pH 8.0)50 µL5Igepal (10% solution)5 µL6Sodium Azide (10% solution)25 µL7H2Oto 5 mL8Total5 ml
The Bead Binding Buffer (2x) can be stored at   for up to six months.
?. [Library Double Size Selection]
Add  Buffer EB to Index PCR
25 µl
?. [Exonuclease I Treatment]
Mix each sample (20 µL per well) with  of Cleanup Beads (22% PEG) for a 1:0.8 ration
16 µl
?. [Exonuclease I Treatment]
Incubate for  at  to allow binding of the cDNA onto beads
20 Room temperature
?. [Exonuclease I Treatment]
Place the tube on the magnet stand until clear (~3 min) and discard supernatant
?. [Exonuclease I Treatment]
Wash with  of 80% EtOH while the tube is on the magnet. Discard the supernatant
50 µl
?. [Exonuclease I Treatment]
Repeat wash step once more
?. [Exonuclease I Treatment]
Air dry beads for
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
?. [Exonuclease I Treatment]
Elute the beads in  of UltraPure Water
20 µl
?. [Library Preparation]
Before starting, read the library preparation section carefully as there are a few steps that are very time sensitive.
?. [DNAse I Digest]
Add  H2O and resuspend beads by vortexing vigorously
5 µl
If you encounter bead clumping at this step, try to resuspend the beads by vigorously pipetting the samples. We have generated high quality prime-seq libraries despite heavy clumping.
?. [cDNA Bead Purification ]
Incubate for   at RT and transfer to a new PCR tube or plate
Stopping Point. Samples can be safely stored at   and protocol can be continued at a later date.
?. [cDNA Pooling & Purification]
Incubate for   at RT and transfer to a new PCR tube or plate
?. [Exonuclease I Treatment]
Incubate for   at RT and transfer to a new PCR tube or plate
?. [Preparation]
Thaw frozen buffers and primers on ice.
?. [Preparation]
Prepare 80% EtOH (approximately 45 mL for 96 samples)
?. [Reverse Transcription]
Add  H2O
4 µl
The 4 µL of  water can be combined with the Reverse Transcription Mix by increasing the water in Row 6 from 2.35 µL to 6.35 µL. If working with many samples, or if using a stepper pipette or robot, we find that it is better to add some water separately to prevent the beads from drying too much.
?. [cDNA Pooling & Purification]
Place the plate on a magnet
?. [Adapter Ligation]
Add  Buffer EB to Samples
37.3 µl
?. [Adapter Ligation]
Mix Index PCR with  SPRI select beads (ratio of 1:0.52)
26 µl
We use SPRI Select Beads here instead of our home made 22% Clean Up beads for their guaranteed QCed size selection properties.
?. [Adapter Ligation]
Incubate for  at
[(Room Temp)]
?. [Adapter Ligation]
Place the plate on the magnet stand until clear and transfer 76 µl supernatant to clean well.
Be careful not to discard! This is your sample!
?. [Adapter Ligation]
Mix supernatant with  SPRI select beads (ratio of 1:0.72)
10 µl
?. [Adapter Ligation]
Incubate for  at
[(Room Temp)]
?. [Adapter Ligation]
Place the plate on the magnet stand until clear and discard supernatant.
?. [Adapter Ligation]
Wash with  of 80% EtOH while the plate is on the magnet. Discard the supernatant
150 µl
?. [Adapter Ligation]
Repeat wash step once more
?. [Adapter Ligation]
Air dry beads for
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
?. [Sample Collection]
Prepare Lysis Buffer according to the number of samples.  AB1ReagentTube2RLT Plus Buffer270 µL3β-mercaptoethanol30 µL4Total
300 µL
AB1ReagentTube2RLT Plus Buffer270 µL3β-mercaptoethanol30 µL4Total
300 µL
?. [Sample Collection]
Add of Lysis Buffer to a 2 mL tube.
300 µl
This step can be adjusted based on which tissue homogenizer you are using. We have successfully used the Qiagen Tissue Lyzer, which works well with 300 µL of lysate.
?. [Sample Collection]
Weigh the tissue and add to tube.
?. [Sample Collection]
Dilute the homogenized lysate with additional Lysis Buffer for a total of . Ideally, there should be roughly 100-500 ng of RNA in each sample.
100 µl
We have provided a rough guide below for a variety of tissues. However, it is best to optimize this step and perform a qPCR to determine the amount of RNA present in your samples.   AB1TissueWeight per 100 µL (mg)2Amygdala33Thalamus34Midbrain35Cerebellum36Pons37Medulla38Heart29Lung210Spleen111Small Intestines112Colon113Liver114Kidney115Bladder316Testes2
?. [RNA Extraction]
Add  Isopropanol
135 µl
?. [RNA Extraction]
Shake for  at  at 1400 RPM
20 °C
?. [DNase I Digestion]
Prepare DNase I Mix   AB1ReagentWell2DNase I0.5 µL3DNase I Buffer (10x)1 µL4H2O0.5 µL5Total2 µL
AB1ReagentWell2DNase I0.5 µL3DNase I Buffer (10x)1 µL4H2O0.5 µL5Total2 µL
?. [DNase I Digestion]
Add  of DNase I Mix to each well
2 µl
?. [DNase I Digestion]
Incubate DNase I Mix and beads for  at
[(Room Temp)]
?. [DNase I Digestion]
Heat inactivate the DNase I by adding  of EDTA (100 mM) and incubating for  at
1 µl
65 °C
?. [DNase I Digestion]
Place plate on magnet stand until clear (~3 min) and discard the supernatant.
?. [DNase I Digestion]
Wash with  of 80% EtOH while the plate is on the magnet. Discard the supernatant
100 µl
?. [DNase I Digestion]
Repeat wash step once more
?. [DNase I Digestion]
Air dry beads for
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
?. [Reverse Transcription]
Prepare Reverse Transcription Mix  ABC1ReagentWellPlate2Maxima H Minus RT0.15 µL16.5 µL3Maxima RT Buffer (5x)2 µL220 µL4dNTPs (25 mM)0.4 µL44 µL5TSO (E5V7NEXT) (100 uM)0.1 µL11 µL6UltraPure Water2.35 µL258.5 µL7Total5 µL550 µL
ABC1ReagentWellPlate2Maxima H Minus RT0.15 µL16.5 µL3Maxima RT Buffer (5x)2 µL220 µL4dNTPs (25 mM)0.4 µL44 µL5TSO (E5V7NEXT) (100 uM)0.1 µL11 µL6UltraPure Water2.35 µL258.5 µL7Total5 µL550 µL
?. [Reverse Transcription]
Add  Reverse Transcription Mix to samples
5 µl
?. [Reverse Transcription]
Add  of Barcoded oligodT (E3V7NEXT) (10 µM) per well
1 µl
?. [Reverse Transcription]
Incubate for  at
42 °C
?. [cDNA Pooling & Purification]
Place the plate on a magnet
?. [cDNA Pooling & Purification]
Pool all wells of one plate into a 2 mL tube
?. [cDNA Pooling & Purification]
Add  of Cleanup Beads (22% PEG) for each sample for a 1:1 ratio (e.g. 240 µL for 24 samples)
10 µl
The EDTA in the Cleanup Beads (22% PEG) will inactivate the RT and make pooling easier due to the color.
?. [cDNA Pooling & Purification]
Incubate for  at  to allow binding of the cDNA onto beads
20 Room temperature
?. [cDNA Pooling & Purification]
Place the tube on the magnet stand until clear (~3 min) and discard supernatant
?. [cDNA Pooling & Purification]
Wash with  of 80% EtOH while the tube is on the magnet. Discard the supernatant
1 mL
Volume of EtOH should be adjusted depending on the number of samples. More samples will require more EtOH to cover the beads completely.
?. [cDNA Pooling & Purification]
Repeat wash step once more
?. [cDNA Pooling & Purification]
Air dry beads for
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
?. [cDNA Pooling & Purification]
Elute the beads in 17 µL of UltraPure Water
?. [cDNA Pooling & Purification]
Incubate for   at RT and transfer to a new PCR tube or plate
?. [Exonuclease I Treatment]
Add  of ExoI Buffer (10x) and  of Exonuclease I. Incubate as follows:  ABC1StepTemperatureTime2Incubation37 C20 min3Heat Inactivation80 C10 min4Storage4 C∞
2 µl
1 µl
ABC1StepTemperatureTime2Incubation37 C20 min3Heat Inactivation80 C10 min4Storage4 C∞
?. [Exonuclease I Treatment]
Mix each sample (20 µL per well) with  of Cleanup Beads (22% PEG) for a 1:0.8 ration
16 µl
?. [Exonuclease I Treatment]
Incubate for  at  to allow binding of the cDNA onto beads
20 Room temperature
?. [Exonuclease I Treatment]
Place the tube on the magnet stand until clear (~3 min) and discard supernatant
?. [Exonuclease I Treatment]
Wash with  of 80% EtOH while the tube is on the magnet. Discard the supernatant
50 µl
?. [Exonuclease I Treatment]
Repeat wash step once more
?. [Exonuclease I Treatment]
Air dry beads for
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
?. [Exonuclease I Treatment]
Elute the beads in  of UltraPure Water
20 µl
?. [Exonuclease I Treatment]
Incubate for   at RT and transfer to a new PCR tube or plate
?. [Full length cDNA Amplification]
Prepare Pre Amplification Mix  AB1Reagent1x2KAPA HiFi 2x RM25 µL3Pre-amp Primer (SINGV6) (10 uM)3 µL4UltraPure Water2 µL5Total30 µL
AB1Reagent1x2KAPA HiFi 2x RM25 µL3Pre-amp Primer (SINGV6) (10 uM)3 µL4UltraPure Water2 µL5Total30 µL
?. [Full length cDNA Amplification]
Add  Pre Amplification Mix to sample
30 µl
?. [Full length cDNA Amplification]
Incubate the Pre Amplification PCR as follows:  ABCD1StepTemperatureTimeCycles2Initial Denaturation98 C3 min1 cycle3Denaturation98 C15 sec10 cycles*4Annealing65 C30 sec5Elongation72 C4 min6Final Elongation72 C10 min1 cycle7Storage4 C∞
ABCD1StepTemperatureTimeCycles2Initial Denaturation98 C3 min1 cycle3Denaturation98 C15 sec10 cycles*4Annealing65 C30 sec5Elongation72 C4 min6Final Elongation72 C10 min1 cycle7Storage4 C∞
Adjust the number of cycles based on input (sample number, cell number or concentration). As a general guide we recommend:  AB1Total RNA InputCycles210 ng16350 ng144100 ng125500 ng1061000 ng9
?. [cDNA Bead Purification]
Mix sample with  Clean Up Beads (22% PEG) for a ratio of 1:0.8
40 µl
?. [cDNA Bead Purification]
Incubate for  at
[(Room Temp)]
?. [cDNA Bead Purification]
Place the tube on the magnet stand until clear (~3 min) and discard supernatant
?. [cDNA Bead Purification]
Wash with  of 80% EtOH while the tube is on the magnet. Discard the supernatant
100 µl
?. [cDNA Bead Purification]
Repeat wash step once more
?. [cDNA Bead Purification]
Air dry beads for
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
?. [cDNA Bead Purification]
Elute cDNA in  UltraPure Water
10 µl
?. [cDNA Bead Purification]
Incubate for   at RT and transfer to a new PCR tube or plate
Stopping Point. Samples can be safely stored at   and protocol can be continued at a later date.
?. [cDNA Quantification and Quality Check]
Quantify the cDNA using the Quant-iT PicoGreen dsDNA assay kit or equivalent Qubit following the manufacturer's protocol. Use 1 μl of clean cDNA for quantification.
?. [cDNA Quantification and Quality Check]
Quality check the cDNA using the Agilent 2100 Bioanalyzer with High Sensitivity DNA Analysis Kits.
Passing the cDNA quality check does not guarantee that the data will be of high quality, however,  if the cDNA fails the quality check it will usually not yield good libraries and will therefore generate lower quality data.
?. [Library Preparation]
Before starting, read the library preparation section carefully as there are a few steps that are very time sensitive.
?. [Library Preparation]
Prepare Fragmentation Mix  AB1Reagent1x2Ultra II FS Reaction Buffer1.4 µL3Ultra II FS Enzyme Mix0.4 µL4cDNA (4-8 ng/µL)2.5 µL5TE1.7 µL6Total6 µL
AB1Reagent1x2Ultra II FS Reaction Buffer1.4 µL3Ultra II FS Enzyme Mix0.4 µL4cDNA (4-8 ng/µL)2.5 µL5TE1.7 µL6Total6 µL
Ensure that the Ultra II FS Reaction Buffer is completely thawed. If a precipitate is seen in the buffer, pipette up and down several times to break it up, and quickly vortex to mix. Place on ice until use.
Vortex the Ultra II FS Enzyme Mix for 5-8 seconds prior to use for optimal performance.
?. [Library Preparation]
Vortex the Fragmentation Mix for and immediately proceed to step 70
?. [Library Preparation]
Incubate the Fragmentation reaction as follows:  ABC1StepTemperatureTime2Pre-Cool4 C∞3Fragmentation37 C5 min4A Tailing and Phosphorylation65 C30 min5Storage4 C∞
ABC1StepTemperatureTime2Pre-Cool4 C∞3Fragmentation37 C5 min4A Tailing and Phosphorylation65 C30 min5Storage4 C∞
Set heated lid to 75° C. Make sure the lid is at the correct temperature before you start the reaction.Skip the first incubation step once you have added your samples.
?. [Adapter Ligation]
Prepare Adapter Ligation Mix  AB1Reagent1x2NEBNext Ultra II Ligation Master Mix6 µL3NEBNext Ligation Enhancer0.2 µL4prime-seq Adapter (1.5 µM)0.5 µL5Total6.7 µL
AB1Reagent1x2NEBNext Ultra II Ligation Master Mix6 µL3NEBNext Ligation Enhancer0.2 µL4prime-seq Adapter (1.5 µM)0.5 µL5Total6.7 µL
?. [Adapter Ligation]
Add   Adapter Ligation Mix to each replicate
6.7 µl
?. [Adapter Ligation]
Incubate for  at
20 °C
Turn off heated lid
?. [Adapter Ligation]
Add  Buffer EB to Samples
37.3 µl
?. [Adapter Ligation]
Mix Index PCR with  SPRI select beads (ratio of 1:0.52)
26 µl
We use SPRI Select Beads here instead of our home made 22% Clean Up beads for their guaranteed QCed size selection properties.
?. [Adapter Ligation]
Incubate for  at
[(Room Temp)]
?. [Adapter Ligation]
Place the plate on the magnet stand until clear and transfer 76 µl supernatant to clean well.
Be careful not to discard! This is your sample!
?. [Adapter Ligation]
Mix supernatant with  SPRI select beads (ratio of 1:0.72)
10 µl
?. [Adapter Ligation]
Incubate for  at
[(Room Temp)]
?. [Adapter Ligation]
Place the plate on the magnet stand until clear and discard supernatant.
?. [Adapter Ligation]
Wash with  of 80% EtOH while the plate is on the magnet. Discard the supernatant
150 µl
?. [Adapter Ligation]
Repeat wash step once more
?. [Adapter Ligation]
Air dry beads for
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
?. [Adapter Ligation]
Elute samples in  0.1X TE (dilute 1X TE Buffer 1:10 in water) for
10.5 µl
?. [Library PCR]
Transfer samples to clean wells
?. [Library PCR]
Add  of Index Primer (i7, 5 uM) to each well
1 µl
This is the unique index that will be used for demultiplexing libraries.
?. [Library PCR]
Prepare Library PCR Mix  ABC1Reagent1x5x2NEBNext Ultra II Q5 Master Mix12.5 µL62.5 µL3P5NEXTPT5 primer (5uM stock)1 µL5 µL4Total13.5 µL67.5 µL
ABC1Reagent1x5x2NEBNext Ultra II Q5 Master Mix12.5 µL62.5 µL3P5NEXTPT5 primer (5uM stock)1 µL5 µL4Total13.5 µL67.5 µL
Although scaled down, there will not be sufficient Q5 Master Mix (M0544L) in the kit. This item will have to be ordered separately.
?. [Library PCR]
Add  of Library PCR Mix to each well
13.5 µl
?. [Library PCR]
Incubate the Library PCR reaction as follows:  ABCD1StepTemperatureTimeCycles2Initial Denaturation98 C30 sec1 cycle3Denaturation98 C10 sec10 cycles*4Annealing/Elongation65 C1 min 15 sec5Final Elongation65 C5 min1 cycle6Storage4 C∞
ABCD1StepTemperatureTimeCycles2Initial Denaturation98 C30 sec1 cycle3Denaturation98 C10 sec10 cycles*4Annealing/Elongation65 C1 min 15 sec5Final Elongation65 C5 min1 cycle6Storage4 C∞
Adjust the number of cycles based on cDNA input.As a general guide we recommend:  AB1cDNA InputCycles220 ng10310 ng1145 ng12
?. [Library Double Size Selection]
Add  Buffer EB to Index PCR
25 µl
?. [Library Double Size Selection]
Mix Index PCR with  SPRI select beads (ratio of 1:0.52)
26 µl
We use SPRI Select Beads here instead of our home made 22% Clean Up beads for their guaranteed QCed size selection properties.
?. [Library Double Size Selection]
Incubate for  at
[(Room Temp)]
?. [Library Double Size Selection]
Place the plate on the magnet stand until clear and transfer  supernatant to clean well.
76 µl
Be careful not to discard! This is your library.
?. [Library Double Size Selection]
Mix supernatant with  SPRI select beads (ratio of 1:0.72)
10 µl
?. [Library Double Size Selection]
Incubate for  at
[(Room Temp)]
?. [Library Double Size Selection]
Place the plate on the magnet stand until clear and discard supernatant.
?. [Library Double Size Selection]
Wash with  of 80% EtOH while the plate is on the magnet. Discard the supernatant
150 µl
?. [Library Double Size Selection]
Repeat wash step once more
?. [Library Double Size Selection]
Air dry beads for
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
?. [Library Double Size Selection]
Elute in  UltraPure Water.
15 µl
?. [Library Double Size Selection]
Incubate for  and then place on magnet until clear. Transfer eluted library to new well.
Stopping point. The libraries can be safely stored at   until they wil be QCed and sequenced.
?. [Library QC]
Quantify and quality control the library using the Agilent 2100 Bioanalyzer with High Sensitivity DNA Analysis Kits.
Bulk libraries often yield high concentrations, which should be diluted to get accurate molarity measurements on the Bioanalyzer. Ideally, do not load more than 2 ng onto the chip.
Successful libraries will typically exceed 3-5 ng/μl concentration
?. [Sequencing]
Samples should be submitted according to your Sequencing Facility specifications. At least 8 cycles are required for the Index Read (i7) and 28 cycles for the Read 1 (BC+UMI). Read 2 (DNA) should be adjusted based on the quality of the genome being mapped to, but for human and mouse 50 cycles is sufficient.Some potential sequencing options:  ABCDEF1ReadRead 1Read 2Index Read (i7)Index Read (i5)Kit2HiSeq2811480HiSeq 3000/4000 150 cycles3NextSeq285680NextSeq 500/550 HiOut v3 75 cycle4NovaSeq289488NovaSeq SP v1.5 100 cycle
ABCDEF1ReadRead 1Read 2Index Read (i7)Index Read (i5)Kit2HiSeq2811480HiSeq 3000/4000 150 cycles3NextSeq285680NextSeq 500/550 HiOut v3 75 cycle4NovaSeq289488NovaSeq SP v1.5 100 cycle
?. [Sample Collection]
Homogenize the tissue, while making sure to keep the samples cold
We have successfully used the Qiagen Tissue Lyzer, at 50 oscillation for 2 min for tougher tissue and 30 oscillation for 2 min for softer tissue.
?. [Reverse Transcription]
Prepare Reverse Transcription Mix  ABC1ReagentWellPlate2Maxima H Minus RT0.15 µL16.5 µL3Maxima RT Buffer (5x)2 µL220 µL4dNTPs (25 mM)0.4 µL44 µL5TSO (E5V7NEXT) (100 uM)0.1 µL11 µL6UltraPure Water2.35 µL258.5 µL7Total5 µL550 µL
ABC1ReagentWellPlate2Maxima H Minus RT0.15 µL16.5 µL3Maxima RT Buffer (5x)2 µL220 µL4dNTPs (25 mM)0.4 µL44 µL5TSO (E5V7NEXT) (100 uM)0.1 µL11 µL6UltraPure Water2.35 µL258.5 µL7Total5 µL550 µL
?. [Reverse Transcription]
Add  H2O
4 µl
The 4 µL of  water can be combined with the Reverse Transcription Mix by increasing the water in Row 6 from 2.35 µL to 6.35 µL. If working with many samples, or if using a stepper pipette or robot, we find that it is better to add some water separately to prevent the beads from drying too much.
?. [Reverse Transcription]
Add  Reverse Transcription Mix
5 µl
?. [Reverse Transcription]
Add  of Barcoded oligodT (E3V7NEXT) (10 µM) per well
1 µl
?. [Reverse Transcription]
Incubate for  at
42 °C
?. [cDNA Pooling & Purification]
Place the plate on a magnet
?. [cDNA Pooling & Purification]
Pool all wells of one plate into a 2 mL tube
?. [cDNA Pooling & Purification]
Add  of Cleanup Beads (22% PEG) for each sample for a 1:1 ratio (e.g. 240 µL for 24 samples)
10 µl
The EDTA in the Cleanup Beads (22% PEG) will inactivate the RT and make pooling easier due to the color.
?. [cDNA Pooling & Purification]
Incubate for  at  to allow binding of the cDNA onto beads
20 Room temperature
?. [cDNA Pooling & Purification]
Wash with  of 80% EtOH while the tube is on the magnet. Discard the supernatant
1 mL
Volume of EtOH should be adjusted depending on the number of samples. More samples will require more EtOH to cover the beads completely.
?. [cDNA Pooling & Purification]
Repeat wash step once more
?. [cDNA Pooling & Purification]
Air dry beads for
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
?. [cDNA Pooling & Purification]
Elute the beads in 17 µL of UltraPure Water
?. [cDNA Pooling & Purification]
Incubate for   at RT and transfer to a new PCR tube or plate
?. [Exonuclease I Treatment]
Add  of ExoI Buffer (10x) and  of Exonuclease I. Incubate as follows:  ABC1StepTemperatureTime2Incubation37 C20 min3Heat Inactivation80 C10 min4Storage4 C∞
2 µl
1 µl
ABC1StepTemperatureTime2Incubation37 C20 min3Heat Inactivation80 C10 min4Storage4 C∞
?. [Exonuclease I Treatment]
Mix each sample (20 µL per well) with  of Cleanup Beads (22% PEG) for a 1:0.8 ration
16 µl
?. [Exonuclease I Treatment]
Incubate for  at  to allow binding of the cDNA onto beads
20 Room temperature
?. [Exonuclease I Treatment]
Place the tube on the magnet stand until clear (~3 min) and discard supernatant
?. [Exonuclease I Treatment]
Wash with  of 80% EtOH while the tube is on the magnet. Discard the supernatant
50 µl
?. [Exonuclease I Treatment]
Repeat wash step once more
?. [Exonuclease I Treatment]
Air dry beads for
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
?. [Exonuclease I Treatment]
Elute the beads in  of UltraPure Water
20 µl
?. [Exonuclease I Treatment]
Incubate for   at RT and transfer to a new PCR tube or plate
?. [Full length cDNA Amplification]
Prepare Pre Amplification Mix  AB1Reagent1x2KAPA HiFi 2x RM25 µL3Pre-amp Primer (SINGV6) (10 uM)3 µL4UltraPure Water2 µL5Total30 µL
AB1Reagent1x2KAPA HiFi 2x RM25 µL3Pre-amp Primer (SINGV6) (10 uM)3 µL4UltraPure Water2 µL5Total30 µL
?. [Full length cDNA Amplification]
Add  Pre Amplification Mix to sample
30 µl
?. [Full length cDNA Amplification]
Incubate the Pre Amplification PCR as follows:  ABCD1StepTemperatureTimeCycles2Initial Denaturation98 C3 min1 cycle3Denaturation98 C15 sec10 cycles*4Annealing65 C30 sec5Elongation72 C4 min6Final Elongation72 C10 min1 cycle7Storage4 C∞
ABCD1StepTemperatureTimeCycles2Initial Denaturation98 C3 min1 cycle3Denaturation98 C15 sec10 cycles*4Annealing65 C30 sec5Elongation72 C4 min6Final Elongation72 C10 min1 cycle7Storage4 C∞
Adjust the number of cycles based on input (sample number, cell number, or concentration). As a general guide we recommend:  AB1Total RNA InputCycles210 ng16350 ng144100 ng125500 ng1061000 ng9
?. [cDNA Bead Purification]
Mix sample with  Clean Up Beads (22% PEG) for a ratio of 1:0.8
40 µl
?. [cDNA Bead Purification]
Incubate for  at
[(Room Temp)]
?. [cDNA Bead Purification]
Place the tube on the magnet stand until clear (~3 min) and discard supernatant
?. [cDNA Bead Purification]
Wash with  of 80% EtOH while the tube is on the magnet. Discard the supernatant
100 µl
?. [cDNA Bead Purification]
Repeat wash step once more
?. [cDNA Bead Purification]
Air dry beads for
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
?. [cDNA Bead Purification]
Elute cDNA in  UltraPure Water
10 µl
?. [cDNA Bead Purification]
Incubate for   at RT and transfer to a new PCR tube or plate
Stopping Point. Samples can be safely stored at   and protocol can be continued at a later date.
?. [cDNA Quantification and Quality Check]
Quantify the cDNA using the Quant-iT PicoGreen dsDNA assay kit or equivalent Qubit following the manufacturer's protocol. Use 1 μl of clean cDNA for quantification.
?. [cDNA Quantification and Quality Check]
Quality check the cDNA using the Agilent 2100 Bioanalyzer with High Sensitivity DNA Analysis Kits.
Passing the cDNA quality check does not guarantee that the data will be of high quality, however,  if the cDNA fails the quality check it will usually not yield good libraries and will therefore generate lower quality data.
?. [Library Preparation]
Before starting, read the library preparation section carefully as there are a few steps that are very time sensitive.
?. [Library Preparation]
Prepare Fragmentation Mix  AB1Reagent1x2Ultra II FS Reaction Buffer1.4 µL3Ultra II FS Enzyme Mix0.4 µL4cDNA (4-8 ng/µL)2.5 µL5TE1.7 µL6Total6 µL
AB1Reagent1x2Ultra II FS Reaction Buffer1.4 µL3Ultra II FS Enzyme Mix0.4 µL4cDNA (4-8 ng/µL)2.5 µL5TE1.7 µL6Total6 µL
Ensure that the Ultra II FS Reaction Buffer is completely thawed. If a precipitate is seen in the buffer, pipette up and down several times to break it up, and quickly vortex to mix. Place on ice until use.
Vortex the Ultra II FS Enzyme Mix for 5-8 seconds prior to use for optimal performance.
?. [Library Preparation]
Vortex the Fragmentation Mix for and immediately proceed to step 70
?. [Library Preparation]
Incubate the Fragmentation reaction as follows:  ABC1StepTemperatureTime2Pre-Cool4 C∞3Fragmentation37 C5 min4A Tailing and Phosphorylation65 C30 min5Storage4 C∞
ABC1StepTemperatureTime2Pre-Cool4 C∞3Fragmentation37 C5 min4A Tailing and Phosphorylation65 C30 min5Storage4 C∞
Set heated lid to 75° C. Make sure the lid is at the correct temperature before you start the reaction.Skip the first incubation step once you have added your samples.
?. [Adapter Ligation]
Prepare Adapter Ligation Mix  AB1Reagent1x2NEBNext Ultra II Ligation Master Mix6 µL3NEBNext Ligation Enhancer0.2 µL4prime-seq Adapter (1.5 µM)0.5 µL5Total6.7 µL
AB1Reagent1x2NEBNext Ultra II Ligation Master Mix6 µL3NEBNext Ligation Enhancer0.2 µL4prime-seq Adapter (1.5 µM)0.5 µL5Total6.7 µL
?. [Adapter Ligation]
Add   Adapter Ligation Mix to each replicate
6.7 µl
?. [Adapter Ligation]
Incubate for  at
20 °C
Turn off heated lid
?. [Adapter Ligation]
Add  Buffer EB to Samples
37.3 µl
?. [Adapter Ligation]
Mix Index PCR with  SPRI select beads (ratio of 1:0.52)
26 µl
We use SPRI Select Beads here instead of our home made 22% Clean Up beads for their guaranteed QCed size selection properties.
?. [Adapter Ligation]
Incubate for  at
[(Room Temp)]
?. [Adapter Ligation]
Place the plate on the magnet stand until clear and transfer 76 µl supernatant to clean well.
Be careful not to discard! This is your sample!
?. [Adapter Ligation]
Mix supernatant with  SPRI select beads (ratio of 1:0.72)
10 µl
?. [Adapter Ligation]
Incubate for  at
[(Room Temp)]
?. [Adapter Ligation]
Place the plate on the magnet stand until clear and discard supernatant.
?. [Adapter Ligation]
Wash with  of 80% EtOH while the plate is on the magnet. Discard the supernatant
150 µl
?. [Adapter Ligation]
Repeat wash step once more
?. [Adapter Ligation]
Air dry beads for
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
?. [Adapter Ligation]
Elute samples in  0.1X TE (dilute 1X TE Buffer 1:10 in water) for
10.5 µl
?. [Library PCR]
Transfer samples to clean wells
?. [Library PCR]
Add  of Index Primer (i7, 5 uM) to each well
1 µl
This is the unique index that will be used for demultiplexing libraries.
?. [Library PCR]
Prepare Library PCR Mix  ABC1Reagent1x5x2NEBNext Ultra II Q5 Master Mix12.5 µL62.5 µL3P5NEXTPT5 primer (5uM stock)1 µL5 µL4Total13.5 µL67.5 µL
ABC1Reagent1x5x2NEBNext Ultra II Q5 Master Mix12.5 µL62.5 µL3P5NEXTPT5 primer (5uM stock)1 µL5 µL4Total13.5 µL67.5 µL
Although scaled down, there will not be sufficient Q5 Master Mix (M0544L) in the kit. This item will have to be ordered separately.
Conversely, one can use unique i5 Indices for each sample. This is recommended if using a sequencer with patterned flow cells due to index hopping.
?. [Library PCR]
Add  of Library PCR Mix to each well
13.5 µl
?. [Library PCR]
Incubate the Library PCR reaction as follows:  ABCD1StepTemperatureTimeCycles2Initial Denaturation98 C30 sec1 cycle3Denaturation98 C10 sec10 cycles*4Annealing/Elongation65 C1 min 15 sec5Final Elongation65 C5 min1 cycle6Storage4 C∞
ABCD1StepTemperatureTimeCycles2Initial Denaturation98 C30 sec1 cycle3Denaturation98 C10 sec10 cycles*4Annealing/Elongation65 C1 min 15 sec5Final Elongation65 C5 min1 cycle6Storage4 C∞
Adjust the number of cycles based on cDNA input.As a general guide we recommend:  AB1cDNA InputCycles220 ng10310 ng1145 ng12
?. [Library Double Size Selection]
Add  Buffer EB to Index PCR
25 µl
?. [Library Double Size Selection]
Mix Index PCR with  SPRI select beads (ratio of 1:0.52)
26 µl
We use SPRI Select Beads here instead of our home made 22% Clean Up beads for their guaranteed QCed size selection properties.
?. [Library Double Size Selection]
Incubate for  at
[(Room Temp)]
?. [Library Double Size Selection]
Place the plate on the magnet stand until clear and transfer  supernatant to clean well.
76 µl
Be careful not to discard! This is your library.
?. [Library Double Size Selection]
Mix supernatant with  SPRI select beads (ratio of 1:0.72)
10 µl
?. [Library Double Size Selection]
Incubate for  at
[(Room Temp)]
?. [Library Double Size Selection]
Place the plate on the magnet stand until clear and discard supernatant.
?. [Library Double Size Selection]
Wash with  of 80% EtOH while the plate is on the magnet. Discard the supernatant
150 µl
?. [Library Double Size Selection]
Repeat wash step once more
?. [Library Double Size Selection]
Air dry beads for
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
?. [Library Double Size Selection]
Elute in  UltraPure Water.
15 µl
?. [Library Double Size Selection]
Incubate for  and then place on magnet until clear. Transfer eluted library to new well.
Stopping point. The libraries can be safely stored at   until they wil be QCed and sequenced.
?. [Library QC]
Quantify and quality control the library using the Agilent 2100 Bioanalyzer with High Sensitivity DNA Analysis Kits.
Bulk libraries often yield high concentrations, which should be diluted to get accurate molarity measurements on the Bioanalyzer. Ideally, do not load more than 2 ng onto the chip.
Successful libraries will typically be 2-5 ng/μl concentration
?. [Sequencing]
Samples should be submitted according to your Sequencing Facility specifications. At least 8 cycles are required for the Index Read (i7) and 28 cycles for the Read 1 (BC+UMI). Read 2 (DNA) should be adjusted based on the quality of the genome being mapped to, but for human and mouse 50 cycles is sufficient.Some potential sequencing options:  ABCDEF1ReadRead 1Read 2Index Read (i7)Index Read (i5)Kit2HiSeq2811480HiSeq 3000/4000 150 cycles3NextSeq285680NextSeq 500/550 HiOut v3 75 cycle4NovaSeq289488NovaSeq SP v1.5 100 cycle
ABCDEF1ReadRead 1Read 2Index Read (i7)Index Read (i5)Kit2HiSeq2811480HiSeq 3000/4000 150 cycles3NextSeq285680NextSeq 500/550 HiOut v3 75 cycle4NovaSeq289488NovaSeq SP v1.5 100 cycle
?. [RNA Extraction]
Add  Cleanup Beads (22% PEG)
70 µl
?. [RNA Extraction]
Shake for  at  at 1400 RPM
20 °C
?. [RNA Extraction]
Place on magnet stand until clear (~3 min) and then discard supernatant
?. [RNA Extraction]
Add  Isopropanol
300 µl
?. [RNA Extraction]
Shake for  at  at 1400 RPM
20 °C
?. [RNA Extraction]
Place on magnet stand until clear (~3 min) and then discard supernatant
?. [RNA Extraction]
Wash with  of 80% EtOH while the plate is on the magnet. Discard the supernatant
300 µl
?. [RNA Extraction]
Repeat wash step once more
Drying can be controlled better if the tubes are briefly spun down before aspirating the supernatant after the second wash.
?. [RNA Extraction]
Air dry beads for
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
?. [RNA Extraction]
Elute Extracted RNA with   H2O
20 µl
?. [RNA Extraction]
Shake for  at  at 1400 RPM
20 °C
?. [RNA Quantification]
Quantify the cDNA using the Quantifluor RNA System or equivalent following the manufacturer's protocol. Use  of RNA for quantification.
1 µl
?. [DNase I Digestion]
Transfer  of Extracted RNA to 96-well plate.
8 µl
?. [DNase I Digestion]
Elute RNA with   H2O
15 µl
?. [DNase I Digestion]
Incubate for   at RT and transfer to a new PCR tube or plate
Stopping Point: Extracted RNA can be stored at -80 °C until you are ready to proceed to the next steps.
?. [RNA Quantification]
Normalize the samples so they are all the same concentration.
Minimum: 4 ng (1 ng/µL)Maximum: 40 ng (10 ng/µL)
?. [RNA Quantification]
Transfer  of the normalized RNA to a new plate. Return the unnormalized RNA to the freezer.
4 µl
?. [Sample Preparation]
Normalize the samples so they are all the same concentration.
Minimum: 4 ng (1 ng/µL)Maximum: 40 ng (10 ng/µL)
?. [Sample Preparation]
Transfer  of the normalized RNA to a new plate. Return the unnormalized RNA to the freezer.
4 µl