TITLE: NASC-seq (new transcriptome alkylation-dependent single-cell RNA sequencing) protocol
AUTHORS: Gert-Jan Hendriks, Lisa Anna Jung, Anton J.M. Larsson, Oscar Andersson Forsman, Michael Lidschreiber, Katja Lidschreiber, Patrick Cramer, Rickard Sandberg

[DESCRIPTION]
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[STEPS]
?. [Prepare lysis plate]
Prepare the following lysis buffer on ice.  ABC1Reagent1 reaction96-well plate2Sodium Phosphate buffer (200mM)2.5 μl264 μl3RNAse inhibitor0.2 μl21.12 μl4ERCC + 4sU Spike-in mix0.1 μl10.56 μl5Nuclease-free water0.2 μl21.12 μl6Total3 μl316.8 μl
ABC1Reagent1 reaction96-well plate2Sodium Phosphate buffer (200mM)2.5 μl264 μl3RNAse inhibitor0.2 μl21.12 μl4ERCC + 4sU Spike-in mix0.1 μl10.56 μl5Nuclease-free water0.2 μl21.12 μl6Total3 μl316.8 μl
[Keep lysis mix on ice.]
?. [Prepare lysis plate]
Distribute 3 μl of freshly prepared lysis buffer to each well of a 96-well PCR plate using a multichannel or high-throughput liquid dispensing system.
[Distribute lysis mix.]
[Keep lysis plate on ice. ]
?. [Cell culture and FACS sorting]
Distribute single cells into each well of the lysis plate by FACS sorting.
Seal the  plates with aluminium foil seal and store at -80°C.
[Keep cells and lysis plate cooled before, during and after sorting.]
[Store lysis plate.]
?. [Oligo-dT bead preparation]
Transfer 60 μl Dynabeads to a clean eppendorf tube (for a 96-well plate).
Dynabeads sediment with time and need to be vortexed well before pipetting.Do not use DNA-low-bind consumables since dynabeads may stick to the tips and tubes
?. [Alkylation]
Prepare the alkylation mix at room temperature.  ABC1Reagent1 reaction96-well plate2DMSO4.5 μl517.5 μl3Iodoacetamide (200mM, in DMSO)0.5 μl57.5 μl4Total5 μl575 μl
ABC1Reagent1 reaction96-well plate2DMSO4.5 μl517.5 μl3Iodoacetamide (200mM, in DMSO)0.5 μl57.5 μl4Total5 μl575 μl
[Keep at room temperature]
To avoid problems with iodocatamide stability and variability of alkylating potential, we use single-use aliquots of iodoacetamide that are dissolved in DMSO at 200mM right before they are used to prepare the alkylation mix above and the remainder is discarded.
Iodoacetamide should be handled in a fume hood and in accordence with local environmental health and safety regulations.
?. [Reverse Transcription]
Remove the supernatant from each well carefully while the plate is on the magnet. Avoid touching the beads. Immediately after pipetting off the supernatant in a column of wells, add 10 ul of Reverse Transcription mix to those wells.
Iodoacetamide should be handled in a fume hood and in accordence with local environmental health and safety regulations.
Avoid touching the beads while pipetting the supernatant off, and pipette the Reverse Transcription mix to the side where the beads are immobilized to avoid drying out the beads.
?. [Binding of beads to RNA]
Remove the lysis plate from -80°C and thaw on ice.
[Thaw lysis plate on ice]
If proceeding directly from FACS sorting the cells to the prepration of the NASC-seq libraries, it is recommended to freeze the cells at -80°C for 5 minutes and continue with step 5 to ensure that the cells are completely lysed.
?. [Prepare Sodium Phosphate buffer]
10 µl
?. [Binding of beads to RNA]
Place the lysis plate in a thermocycler and denature the RNA at 80°C for 3 minutes.
[Lyse cells and denature RNA]
[Cool plate after denaturing RNA]
?. [Binding of beads to RNA]
Distribute 2ul of beads to each well of the lysis plate.
[Oligo-dT beads]
?. [Binding of beads to RNA]
Incubate the beads for 20 minutes at room temperature with agitation.
[Room temperature]
[Bind mRNA to oligo-dT beads]
?. [Alkylation]
Spin the plate down genlty and place on the magnet.Pipette 10 μl of STOP mix to each well of the plate while the plate is on the magnet.
[STOP mix]
Iodoacetamide should be handled in a fume hood and in accordence with local environmental health and safety regulations.
?. [Alkylation]
Spin the plate down gently and place back on the magnet and incubate for 5 minutes at room temperature.
[Room temperature]
Iodoacetamide should be handled in a fume hood and in accordence with local environmental health and safety regulations.
?. [Alkylation]
Add 5  μl of the alkylation mix to each well of the sample plate, spin gently, and incubate with agitation at 50°C for 15 minutes.While the alklylation reaction is running, prepare the STOP mix:  ABC1Reagent1 reaction96-well plate2Superscript II RT buffer4.0 μl460 μl3Nuclease-free water5.10 μl586.50 μl4Tween 20 (10%)0.3 μl34.50 μl5DTT (1M)0.6 μl69.00 μl6Total10 μl1150 μlWhile the alkylation reaction is running, prepare the reverse transcription mix (see step 14)
[Alkylation mix]
50 °C
ABC1Reagent1 reaction96-well plate2Superscript II RT buffer4.0 μl460 μl3Nuclease-free water5.10 μl586.50 μl4Tween 20 (10%)0.3 μl34.50 μl5DTT (1M)0.6 μl69.00 μl6Total10 μl1150 μl
[Keep STOP mix on ice]
?. [Prepare lysis plate]
Optional: Store lysis plate. Seal plate with aluminium foil and store at -80°C, if not immediately continuing with step 4.
[Store lysis plate.]
?. [Pre-amplification PCR]
Add 12 μl of the PCR mix to each well of the plate with the reverse transcribed cDNA.
[PCR mix]
?. [Two-sided AMPure XP cleanup]
Add 18 μl AMPure XP beads to each well of the amplified cDNA libraries and mix by pipetting up and down. Incubate for 15 minutes at room temperature.
[AMPure XP beads]
[Bind DNA to beads]
?. [Reverse Transcription]
Prepare the reverse transcription mix:  ABC1Reagent1 reaction96-well plate2Superscript II Reverse Transcriptase0.5 μl55.2 μl3Superscript II RT buffer2 μl220.8 μl4RNAse inhibitor0.2 μl22.08 μl5DTT (0.1M)0.5 μl55.2 μl6Betaine (5M)2 μl220.8 μl7MgCl2 (1M)0.1 μl11.04 μl8Template Switching Oligo (100μM)0.2 μl22.08 μl9dNTPs (25 mM each)0.4 μl44.16 μl10Nuclease-free water4.1 μl452.64 μl11Total10 μl1104 μl
ABC1Reagent1 reaction96-well plate2Superscript II Reverse Transcriptase0.5 μl55.2 μl3Superscript II RT buffer2 μl220.8 μl4RNAse inhibitor0.2 μl22.08 μl5DTT (0.1M)0.5 μl55.2 μl6Betaine (5M)2 μl220.8 μl7MgCl2 (1M)0.1 μl11.04 μl8Template Switching Oligo (100μM)0.2 μl22.08 μl9dNTPs (25 mM each)0.4 μl44.16 μl10Nuclease-free water4.1 μl452.64 μl11Total10 μl1104 μl
?. [Pre-amplification PCR]
Prepare the PCR mix:  ABC1Reagent1 reaction6-well plate2KAPA HiFi HotStart Readymix (2x)11 μl1214 μl3ISPCR primer (10μM)0.3 μl33.12 μl4H2O0.7 μl77.28 μl5Total12 μl1324.8 μl
ABC1Reagent1 reaction6-well plate2KAPA HiFi HotStart Readymix (2x)11 μl1214 μl3ISPCR primer (10μM)0.3 μl33.12 μl4H2O0.7 μl77.28 μl5Total12 μl1324.8 μl
?. [Reverse Transcription]
Spin the beads down gently and resuspend by careful vortexing.
?. [Reverse Transcription]
Perform the RT according to the following program: 10 cycles:------------------------When the RT program is almost done, continue with step 25
42 °C
50 °C
42 °C
[hold]
To optimize cDNA yield it is recommended to vortex the plate every 20 minutes during the RT program to avoid settling of the beads.
?. [Pre-amplification PCR]
Perform PCR according to the following program:22 cycles:------------------------
98 °C
98 °C
67 °C
72 °C
72 °C
[hold]
This is a good point to interupt the protocol and store the unpurified, amplified cDNA library at -20°C for up to 3 days.
?. [Oligo-dT bead preparation]
Place the beads on a magnet and remove the supernatant, then remove the tube from the magnet and resuspend the beads in 60 μl Dynabeads buffer A.
[Repeat once]
?. [Oligo-dT bead preparation]
Place the beads on a magnet and remove the supernatant, then remove the tube from the magnet and resuspend the beads in 60 μl Dynabeads buffer B.
[Repeat once]
?. [Oligo-dT bead preparation]
Place the Dynabeads on a magnet and remove the supernatant, then remove the tube from the magnet and resuspend the beads in 60 μl Dynabeads 2x binding and washing buffer and 60ul of oligo-dT (100μM).Prepare the Dynabead resuspension mix during the binding reaction:  ABC1Reagent1 reaction96-well plate2Nuclease-free water1.64 μl188.6 μl3RNAse inhibitor0.36 μl41.4 μl4Total2 μl230 μl
Mix the beads and oligonucleotides well by pipetting up and down
[Incubate beads with oligo-dT at room temperature with agitation]
ABC1Reagent1 reaction96-well plate2Nuclease-free water1.64 μl188.6 μl3RNAse inhibitor0.36 μl41.4 μl4Total2 μl230 μl
?. [Oligo-dT bead preparation]
Place the Dynabeads on a magnet and remove the supernatant, then remove the tube from the magnet and resuspend the beads in 60 μl Dynabeads 1x binding and washing buffer.
[Repeat once]
?. [Oligo-dT bead preparation]
Resuspend the beads in 220 μl of freshly prepared bead resuspension buffer.
[Dynabead resuspension mix.]
[Keep prepared beads on ice until ready to use]
?. [Tagmentation (Nextera XT - 1/5 volume)]
Prepare the tagmentation mix  ABC1Reagent1 reaction96-well plate2Tagment DNA buffer (TD)2 μl211.2 μl3Amplicon Tagmentation mix (ATM)1 μl105.6 μl4Total3 μl316.8 μl
ABC1Reagent1 reaction96-well plate2Tagment DNA buffer (TD)2 μl211.2 μl3Amplicon Tagmentation mix (ATM)1 μl105.6 μl4Total3 μl316.8 μl
?. [Tagmentation (Nextera XT - 1/5 volume)]
Pipette 1 μl of each well of the diluted cDNA library (200 pg/μl) plate to the plate with the tagmentation mix and incubate the plate at 55C for 5 minutes.
[Diluted cDNA library (200 pg/μl)]
[Tagment for 5 minutes]
?. [Tagmentation (Nextera XT - 1/5 volume)]
Add 3 μl of Tagmentation mix to each well of a new 96-well plate. Keep this plate on ice after dispensing the amplicon tagmentation mix.
[Keep tagmentation mix and plate on ice]
?. [Tagmentation (Nextera XT - 1/5 volume)]
Stop the tagmentation reaction and remove the tagmentation complexes from the DNA by adding 1 μl of NT buffer to each well and incubating for 5 minutes at room temperature.
[NT buffer]
[Remove tagmentation complexes]
[Room temperature]
?. [cDNA QC]
Analyze 1 μl of 11 selected libraries with a Agilent bioanalyzer on a Agilent DNA high-sensitivity chip.
[Purified cDNA library]
?. [Sequencing]
Sequence the NASC-seq library on an Illlumina NextSeq or NovaSeq with standard sequencing primers.
We recommend sequencing paired-end (2x) 150 cycles to increase the number of conversions that are detected in each read.
?. [Two-sided AMPure XP cleanup]
Move plate to magnet and wait up to 5 minutes to immobilize the beads. Remove the supernatant and wash the beads with 50 μl 80% ethanol.
[Immobilize beads]
[80% ethanol]
?. [Two-sided AMPure XP cleanup]
Resuspend beads into 17 μl of nuclease free water and incubate at room temperature for 3 minutes to release DNA.
[Nuclease-free water]
[Release DNA]
?. [Two-sided AMPure XP cleanup]
Move the plate onto the magnet and allow the beads to immobilize for 5 minutes before moving 16 μl of supernatant to a new plate.
[Immobilize beads]
[Purified cDNA library]
?. [Two-sided AMPure XP cleanup]
Remove the supernatant and dry the beads for 5 minutes.
[Dry beads]
?. [Final Library QC]
Analyze 1 μl of the final NASC-seq library on the Agilent Bioanalyzer using a Agilent DNA high sensitivity chip.
[NASC-seq library]
?. [cDNA QC]
Analyze 1 μl of each well of the cDNA library plate by QuantiFluor assay
[Purified cDNA library]
?. [cDNA QC]
Use the concentrations measured in step 32 to prepare a plate with cDNA from all libraries diluted to 200 pg/μl.
?. [Tagmentation (Nextera XT - 1/5 volume)]
Add 2 ul of premixed Illumina N7/N5 compatible indexing primers (1 μM) to each well.
[Premixed index primer mix (1 μM)]
?. [Tagmentation (Nextera XT - 1/5 volume)]
Add 3 μl of Illumina PCR mix (NPM) to each well of the plate and perform PCR according to the following conditions:10 cycles:------------------------
[NPM]
72 °C
95 °C
95 °C
55 °C
72 °C
72 °C
[hold]
?. [Two-sided AMPure XP cleanup]
Pool 2.5  μl from each well into a new 1.5ml eppendorf tube and transfer 200  μl of the mixed pool to another new eppendorf tube.
?. [Two-sided AMPure XP cleanup]
Place the beads on a magnet and allow beads to immobilize for 5 minutes. Then transfer the supernatant to a new 1.5ml eppendorf tube.
[Immobilize beads]
[Supernatant (pooled Library + PEG) ]
?. [Two-sided AMPure XP cleanup]
Add 60 μl of AMPure XP beads to the 300 μl of library and PEG mixture (supernatant from step 44). Mix by pipetting up and down and allow DNA to bind for 15 minutes.
[AMPure XP beads]
[Bind DNA]
?. [Two-sided AMPure XP cleanup]
Place the beads on the magnet and allow 5 minutes to immobilize the beads. Remove the supernatant and wash the immobilized beads with 200 μl 80% ethanol. Repeat the ethanol wash 4 times.
[Immobilize beads]
[80% ethanol (5x)]
?. [Two-sided AMPure XP cleanup]
Allow the beads to dry for 5 minutes.
[Dry beads]
?. [Two-sided AMPure XP cleanup]
Add 100  μl of AMPure XP beads and allow 5 minutes at room temperature to bind very large framgents and exclude them.
[AMPure XP beads]
[Bind largest fragments to beads]
?. [Two-sided AMPure XP cleanup]
Resuspend the beads in 60 μl of nuclease-free water and allow 5 minutes to release the DNA.
[Release DNA]
?. [Two-sided AMPure XP cleanup]
Transfer 55 μl of the final resuspended NASC-seq library to a new 1.5ml eppendorf tube and keep it on ice.
[NASC-seq library]
[Store NASC-seq library on ice]
?. [Final Library QC]
Measure the concentration of the final NASC-seq library using the Qubit DNA HS kit and prepare a 4nM final library for sequencing.
?. [Cell culture and FACS sorting]
Grow cells in the presence of 50 μM 4sU. It can be helpful to collect untreated cells as a control.
4sU is light-sensitive, and direct light (i.e. light in the cell culture hood, etc) should be reduced to a minimum.Do not refreeze leftovers from 4sU aliquot.Labeling times can vary for different celltypes and biological applications, but in general, short labeling times (i.e. less than 30 minutes) may result in poor performance of the grandSLAM mixture model. Depending on your downstream analysis, this may affect your results.
?. [Cell culture and FACS sorting]
Stop the labeling by transferring your cells to 15-ml falcon tubes on ice and wash them with cold PBS.
[Keep cells on ice.]