TITLE: gmcSCRB-seq protocol
AUTHORS: Johannes Bagnoli, Christoph Ziegenhain, Aleksandar Janjic, Lucas Esteban Wange, Beate Vieth, Swati Parekh, Johanna Geuder, Ines Hellmann, Wolfgang Enard

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>gmcSCRB-seq is an alternative lysis protocol to mcSCRB-seq (</span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Publication</span></a><span> and  </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Protocol</span></a><span>). gmcSCRB-seq uses Guanidine Hydrochloride in the lysis buffer and requires an additional cean up step. We recommend using this alternative lysis protocol in cases where cells are difficult to lyse or where the DNA appears degraded following the pre-amplification step. However, we have observed reduced sensitivity with gmcSCRB-seq compared to mcSCRB-seq and would therefore only recommend using it in cases where the latter does not produce high quality libraries.</span></div></div>

[STEPS]
?. [Preparation of lysis plates]
Prepare 96 well plate(s) containing 5 μL Lysis Buffer per well.
Lysis plates should be prepared shortly before use, but can be stored for up to 1 week at room temperature. Prior to use, double check to make sure salts have not fallen out of solution or that the lysis buffer has not evaporated.
[Lysis Buffer]
?. [Preparation of lysis plates]
Prepare Lysis Buffer for the number of plates needed.    AB1Reagent96-well plate28M Guanadine Hydrochloride343.7532-mercaptoethanol5.54NEB HF Phusion buffer (5x)1.15H2O199.656Total
550
Note that 96-well plates are the preferred setup for gmcSCRB-seq, as the well cleanup (Step 9-15) would be more difficult with 384-well plates.
AB1Reagent96-well plate28M Guanadine Hydrochloride343.7532-mercaptoethanol5.54NEB HF Phusion buffer (5x)1.15H2O199.656Total
550
?. [Sample Collection]
Sort 1 cell to each well of a 96 well plate containing 5 μL Lysis Buffer.
?. [Sample Collection]
Immediately seal the plate with an aluminium cold storage seal.
?. [Sample Collection]
In a cooled centrifuge, spin down the plate for 30 sec @ max. speed and place immediately on dry ice.
[Store plates containing single-cell lysates in a -80 °C freezer for up to 6 months.]
?. [Initial Clean-up]
Thaw plates briefly (up to 1 min) at room temperature
?. [Initial Clean-up]
Spin down (30 sec @ 1000 rcf) in a centrifuge pre-cooled to 4 °C.
?. [Initial Clean-up]
Add 10 uL of Clean-up Beads to each well (1:2 ratio).
[Clean-up Beads]
?. [Reverse Transcription]
Prepare Reverse Transcription Mix as follows:  AB1&nbsp;Reagent&nbsp;96-well plate2UltraPure Water88 μL
3PEG 8000 (50 % solution)165 μL4Maxima RT Buffer (5x)
220 μL5dNTPs (25 mM each)44 μL
6TSO E5V6NEXTunblocked (100 μM)22 μL7Maxima H Minus RT (200 U/μl)&nbsp;
11 μL8Total&nbsp;550 μL
AB1&nbsp;Reagent&nbsp;96-well plate2UltraPure Water88 μL
3PEG 8000 (50 % solution)165 μL4Maxima RT Buffer (5x)
220 μL5dNTPs (25 mM each)44 μL
6TSO E5V6NEXTunblocked (100 μM)22 μL7Maxima H Minus RT (200 U/μl)&nbsp;
11 μL8Total&nbsp;550 μL
- RT MM can be prepared while plate is incubating or while drying on magnet. - If ERCCs will be used, decrease the amount of H2O and add appropriate amount of ERCCs.- Caution: Reverse Transcription Mix with PEG needs to be mixed carefully!
[Keep Reverse Transcription Mix on ice]
?. [Reverse Transcription]
Add 5 μL Reverse Transcription Mix to each well.
[Reverse Transcription Mix]
If a robot (eg. Formulatrix Mantis) is used, make sure to calibrate correctly to the viscous solution.
?. [Reverse Transcription]
Seal plate with a PCR seal, vortex briefly and spin down (30 sec @ 1000 rcf) in a centrifuge pre-cooled to 4 °C.
?. [Reverse Transcription]
In a thermocycler with heated lid, incubate:
[90 min]
[∞]
?. [cDNA Pooling & Purification]
Pool all wells (including beads) of one plate into a 2 mL tube and add 960 μL (ratio 1/1) Bead Binding Buffer
[Bead Binding Buffer]
Vortex to mix the sample and bead binding buffer.
?. [cDNA Pooling & Purification]
[binding of the cDNA onto the beads]
[Room temperature]
?. [cDNA Pooling & Purification]
Place on magnet stand until clear
?. [cDNA Pooling & Purification]
Discard supernatant
?. [cDNA Pooling & Purification]
Wash twice with 2 mL 80% ethanol (while on magnet) and discard supernatant
[80% ethanol (freshly prepared)]
?. [cDNA Pooling & Purification]
[air dry beads]
?. [Exonuclease I Treatment]
Elute cDNA in 17 μL UltraPure Water & transfer to new tube
[UltraPure Water]
Avoid transfering beads as these can inhibit the downstream PCR.
?. [Exonuclease I Treatment]
To the 17 μl cDNA, add:
[Exonuclease I Buffer (10x)]
[Exonuclease I (20 U/μl)]
?. [Full length cDNA amplification]
In a thermocycler with heated lid, incubate:
[20 min (ExoI digest)]
[10 min (Heat inactivation)]
[∞ (Store)]
?. [Full length cDNA amplification]
Prepare PreAmplification Mix as follows:  AB1Reagent1x2Terra direct Buffer (2x)
25&nbsp;μL3SINGV6 Primer (10 μM)
1&nbsp;μL
4Terra polymerase (1.25 U/μL)
1 μL
5UltraPure Water
3&nbsp;μL
6Total
30 μL
AB1Reagent1x2Terra direct Buffer (2x)
25&nbsp;μL3SINGV6 Primer (10 μM)
1&nbsp;μL
4Terra polymerase (1.25 U/μL)
1 μL
5UltraPure Water
3&nbsp;μL
6Total
30 μL
?. [Full length cDNA amplification]
Add 30 µL of PreAmplification Mix directly to the Exonuclease I digested sample.
[PreAmplification Mix]
?. [cDNA purification & quantification]
In a thermocycler with heated lead, incubate as follows:  ABCD1StepTemperatureTimeCycles2Initial Denaturation98 °C3 min1x3Denaturation98&nbsp;°C15 sec&nbsp;
&nbsp;13-21x
4Annealing65&nbsp;°C30 sec5Elongation68&nbsp;°C4 min6Final Elongation72&nbsp;°C10 min&nbsp;
&nbsp;1x
7Store8&nbsp;°C∞
ABCD1StepTemperatureTimeCycles2Initial Denaturation98 °C3 min1x3Denaturation98&nbsp;°C15 sec&nbsp;
&nbsp;13-21x
4Annealing65&nbsp;°C30 sec5Elongation68&nbsp;°C4 min6Final Elongation72&nbsp;°C10 min&nbsp;
&nbsp;1x
7Store8&nbsp;°C∞
Cylce number highly depends on the input amount and should be optimized depending on the specific celltype used in the experiment. For ES cells, 13-15 cycles are sufficient.
?. [Initial Clean-up]
Prepare Clean-up Beads:  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 -Add all ingredients into a 50 mL falcon tube, but do not add the total amount of water until after PEG is completely solubilized
 -Incubate at 40°C and vortex regularly until PEG is completely dissolved
- Resuspend bead stock carefully (Sera-Mag Speed Beads)
- Pipette 1000 μL of bead suspension into a 1.5 mL tube
- Place on magnet stand
 -Remove supernatant
 -Add 1000 μL 10 mM Tris-HCl, pH 8.0, 1 mM EDTA (TE), and resuspend beads
 -Place on magnet stand
 -Remove supernatant
 -Repeat wash one more time
 -Add 900 μL 10 mM Tris-HCl, pH 8.0, 1 mM EDTA (TE), and resuspend beads
 -Add to PEG solution above and mix well.
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
Beads should be prepared ahead of time and can be stored at 4 °C or room temperature.
?. [cDNA purification & quantification]
Mix PreAmplification PCR with 40 μL Clean-up Beads (1/0.8 ratio)
[Clean-up Beads]
?. [cDNA purification & quantification]
[binding of the cDNA onto the beads]
?. [cDNA purification & quantification]
Place on magnet until clear and discard supernatant
?. [cDNA purification & quantification]
Wash twice with 150 µL 80% ethanol (while on magnet) and discard supernatant
[80% ethanol (freshly prepared)]
?. [cDNA purification & quantification]
[air dry beads]
?. [cDNA purification & quantification]
Elute cDNA in 15 μL UltraPure Water & transfer to new tube
[UltraPure Water]
?. [cDNA purification & quantification]
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 concentration should be > 1 ng/μl, depending on cell type and cycle number
?. [cDNA purification & quantification]
Optional: Quality check the cDNA using the Agilent 2100 Bioanalyzer with High Sensitivity DNA Analysis Kits.
?. [Tagmentation, Library PCR & Indexing]
Prepare Tagmentation Mix and dispense 19 μL to a new 96-well plate.
  
   AB1Reagent1x2Tagment DNA&nbsp;Buffer (2x)
10 μL3Amplicon Tagment Mix (Tn5)
5 μL
4UltraPure Water
4 μL
5Total
19 μL
AB1Reagent1x2Tagment DNA&nbsp;Buffer (2x)
10 μL3Amplicon Tagment Mix (Tn5)
5 μL
4UltraPure Water
4 μL
5Total
19 μL
[Tagmentation Mix]
?. [Tagmentation, Library PCR & Indexing]
Dilute cDNA to 0.8 ng/µL and add 1 µL to each reaction.
[cDNA (0.8 ng/µL)]
?. [Tagmentation, Library PCR & Indexing]
In a thermocycler with heated lid, incubate as follows:
[Tagmentation]
[Tagmentation]
?. [Tagmentation, Library PCR & Indexing]
To stop the reaction, add 5 μL NT buffer to each reaction and mix by pipetting up and down.
[NT Buffer]
[Incubation at room temperature]
?. [Tagmentation, Library PCR & Indexing]
Prepare 3' Enrichment PCR Mix as follows and add 24.5 µL to each tagmentation reaction.  AB1Reagent1x2NPM PCR Mix
15 μL3P5NEXTPT5 (5 μM)
0.5 μL
4UltraPure Water
9 μL
5Total
24.5 μL
AB1Reagent1x2NPM PCR Mix
15 μL3P5NEXTPT5 (5 μM)
0.5 μL
4UltraPure Water
9 μL
5Total
24.5 μL
[3' Enrichment PCR Mix]
?. [Tagmentation, Library PCR & Indexing]
Add 0.5 μL of i7 index primer (5 μM)
[i7 index primer (5 μM)]
?. [Tagmentation, Library PCR & Indexing]
In a thermocycler with heated lead, incubate as follows:  ABCD1StepTemperatureTimeCycles2Gap-fill72 °C3 min&nbsp;&nbsp;1x3Initial Denaturation95 °C30 sec4Denaturation95 °C10 sec&nbsp;
&nbsp;13x
5Annealing55&nbsp;°C30 sec6Elongation72&nbsp;°C1&nbsp;min7Final Elongation72&nbsp;°C5&nbsp;min&nbsp;
&nbsp;1x
8Store8&nbsp;°C∞
ABCD1StepTemperatureTimeCycles2Gap-fill72 °C3 min&nbsp;&nbsp;1x3Initial Denaturation95 °C30 sec4Denaturation95 °C10 sec&nbsp;
&nbsp;13x
5Annealing55&nbsp;°C30 sec6Elongation72&nbsp;°C1&nbsp;min7Final Elongation72&nbsp;°C5&nbsp;min&nbsp;
&nbsp;1x
8Store8&nbsp;°C∞
?. [Tagmentation, Library PCR & Indexing]
Mix Index PCR with 50 μL Clean-up Beads (1/1 ratio)
[Clean-up Beads]
?. [Tagmentation, Library PCR & Indexing]
[binding of DNA onto the beads]
?. [Tagmentation, Library PCR & Indexing]
Place on magnet until clear and discard supernatant
?. [Tagmentation, Library PCR & Indexing]
Wash twice with 150 µL 80% ethanol (while on magnet) and discard supernatant
[80% ethanol (freshly prepared)]
?. [Tagmentation, Library PCR & Indexing]
[air dry beads]
?. [Tagmentation, Library PCR & Indexing]
Elute cDNA in 20 μL UltraPure Water & transfer to new tube
[UltraPure Water]
?. [Size selection]
Load complete library onto an 2% Agarose E-Gel EX and run for 10 minutes.
?. [Size selection]
As soon as the Gel run has finished open the Gel framing using the Gel opening tool
?. [Size selection]
Excise the Library from 300bp to 900bp using a clean scalpel
?. [Size selection]
Gel purify the slice using the Qiagen MinElute Kit following manufacturer's guidelines:Add 450 µL Buffer QGDissolve the gel slice in QG for 10 min @ 42 °CAdd 150 μL Isopropanol to the sample and mix by invertingTransfer sample to spin column and centrifuge at 16 000 x g for 1 minDiscard flow through and add 500 μL Buffer QGCentrifuge at 16 000 x g for 1 min and discard flow throughAdd 700 μL Buffer PECentrifuge at 16 000 x g for 1 min and discard flow throughCentrifuge again at 16 000 x g for 1 min to remove residual ethanolTransfer column to a new 1.5 mL microcentrifuge tubeAdd 20 μL H2O to column and incubate for 1 minCentrifuge at 16 000 x g for 1 min to elute and discard the spin column
The Monarch DNA Gel Extraction Kit (NEB T1020L) can also be used.
?. [Library Quantification]
Quantify and quality control the library using the Agilent 2100 Bioanalyzer with High Sensitivity DNA Analysis Kits.    Load both the library and a 1/10 dilution on two different lanes of the chip.
If cDNA was not quality controlled previously, it is strongly suggested to run cDNA on the same chip.
Successful libraries will typically exceed 3-5 ng/μl concentration.
?. [Library Quantification]
If pooling several libraries, combine equal molar amounts.
?. [Sequencing]
Sequence your library on any compatible Illumina sequencer.Dilute libraries to recommended molarity according to Illumina's recommendations (eg. 2 nM).Select the following paired-end read-length settings:  ABC1ReadCyclesContent2Read 116Cell barcode &amp; UMI3Index 18i7 Index4Index 20&nbsp;5Read 250cDNA fragment
ABC1ReadCyclesContent2Read 116Cell barcode &amp; UMI3Index 18i7 Index4Index 20&nbsp;5Read 250cDNA fragment
?. [Primary data processing using zUMIs]
Download and install zUMIs including all dependencies.
?. [Primary data processing using zUMIs]
Copy the sequencing data from the sequencer and run bcl2fastq without demultiplexing.
?. [Primary data processing using zUMIs]
Run zUMIs by specifying your parameters in the associated yaml file or using the YAML config Rshiny application. Below is an example yaml file for a typical gmcSCRB-seq run.
?. [Initial Clean-up]
Seal the plate and vortex to mix the bead-lysate mixture. Briefly spin down the plate (
?. [Initial Clean-up]
[air dry beads]
Depending on lab temperature and humidity, drying times can vary.
?. [Initial Clean-up]
Wash twice with 50 uL 80% ethanol (while on magnet) and discard supernatant
[80% ethanol (freshly prepared)]
?. [Initial Clean-up]
Discard supernatant
?. [Initial Clean-up]
Place on magnet stand until clear
?. [Initial Clean-up]
Incubate for 5 minutes at RT.
[binding of the cDNA onto the beads]
[Room temperature]
?. [Reverse Transcription]
Once drying is complete, add 4 μL H2O to each well.
[H2O]
?. [Reverse Transcription]
Add 1 μL of barcoded oligo-dT primer [2 μM] (E3V6NEXT adapter) to each well.
[barcoded oligo-dT primer [2 μM] (E3V6NEXT adapter)]
?. [cDNA Pooling & Purification]
Vortex to mix the bead-cDNA mixture. Briefly spin down the plate (
?. [cDNA Pooling & Purification]
Prepare Bead Binding Buffer  AB1ReagentAmount2PEG 800011 g
3NaCl, 5M
10 mL4Tris-HCL, 1M, pH 8.0
500 μL5EDTA, 0.5M
100 μL
6IGEPAL, 10% solution
50 μL7Sodium Azide, 10% solution
250 μL8UltraPure Waterup to 50 mL9Total 50 mL-Add all ingredients into a 50 mL falcon tube, but do not add the total amount of water until after PEG is completely solubilized
 -Incubate at 40°C and vortex regularly until PEG is completely dissolved
AB1ReagentAmount2PEG 800011 g
3NaCl, 5M
10 mL4Tris-HCL, 1M, pH 8.0
500 μL5EDTA, 0.5M
100 μL
6IGEPAL, 10% solution
50 μL7Sodium Azide, 10% solution
250 μL8UltraPure Waterup to 50 mL9Total 50 mL