TITLE: RoCK and ROI: bead modification, library generation and sequencing protocol
AUTHORS: Giulia Moro, Konrad Basler, Erich Brunner

[DESCRIPTION]
Various tools have been developed to reliably identify, trace and analyze single cells in complex tissues. In recent years, these technologies have been combined with transcriptomic profiling approaches to explore molecular mechanisms that drive development, health, and disease. A remaining challenge is that important information relevant for understanding the biology of cells or tissues, such as lowly expressed transcripts, sequence variations or exon junctions, remains undetected. We developed an scRNAseq workflow, RoCK and ROI (Robust Capture of Key transcripts and Region Of Interest), that tackles these limitations. RoCKseq uses targeted capture to enrich for key transcripts, thereby enhancing the detection, identification and tracking of cell types in scRNAseq experiments. ROIseq directs a subset of reads to a specific region of interest via selective priming. This allows specific sequence information to be retrieved for mRNAs of interest, enabling, for example, the inspection of sequence variations. Importantly, the targeted information obtained with RoCK and ROI is recorded together with standard transcriptome readouts. To analyze the multimodal information provided by RoCK and ROI, we developed a novel pipeline. The entire workflow increases the information obtained for lowly expressed genes and enables the detection of individual sequence variations and the exploration of the biological relevance and consequences of the respective variation for the cells expressing it. 

This protocol covers the following steps: 

Design of RoCKseq capture sequences and ROIseq primers 

RoCKseq bead modification on BD Rhapsody beads

RoCK and ROI library generation 

Sequencing of RoCK and ROI libraries

[BEFORE_START]
Important points to keep into consideration during RoCKseq bead modification

LoBind DNA tubes and pipette tips guarantee low bead loss during modification, which otherwise get stuck on walls of pipette tips and tube

Beads should be kept on ice whenever possible

Bead modification should be performed in a clean, RNAse-free hood

Enzymes should be kept at -20°C as long as possible and buffers and splints should be placed on ice after thawing

If multiple samples are processed in parallel, only wash up to four samples at a time to prevent incubation on the magnetic stand for too long

Try limiting (i.e. restrict to 1 minute) the time the beads are exposed to the magnetic stand

Avoid the drying out of the beads after washing

To minimise bead loss during modification: consistently use LoBind DNA Eppendorf tubes and LoBind pipette tips and wait for all the beads to be gathered at the magnet of the magnetic stand before exchanging buffers. During washes ensure that all liquid is expelled from the tip as to minimize bead loss

Important points to keep into consideration during the Fluorescent assay

After addition of the lysis buffer keep beads at room temperature. Do not place back on ice. This may lead to higher fluorescent background signal in the negative control 

The fluorescent probes and the beads with the fluorescent probe should be kept in the dark whenever possible 


[GUIDELINES]
IMPORTANT: This protocol refers to “Enhanced Cell Capture Beads V2” (Part Number: 700034960). For “Enhanced Cell Capture Beads V3” (Part number 91-1294), the sequence of the splint is: 

5’-NNNNNNNNNNNNNNNNNNNNNNNNTATAATCACGACTCCAC-3’ 

[STEPS]
SECTION: Step 1 modification of full vial of RoCKseq beads: preparation of reagents
6. Thaw lambda exonuclease buffer, T4 polymerase buffer, 100 µM splint(s), polyA oligo and 10 mM dNTPs at room temperature and place on ice
SECTION: Step 1 modification of full vial of RoCKseq beads: preparation of reagents
7. Preheat two thermomixers to 75 °C and to37 °C, respectively
SECTION: Step 1 modification of full vial of RoCKseq beads: preparation of reagents
8. Prepare TE/TW and Water buffers in 50 mL Falcons and place on ice 

TE/TW buffer: 500 µL Tris, 100 µL EDTA, 10 µL Tween20, up to 50 mL with ddH2O
Water buffer: 10 µLTween20, up to 50 mL with ddH2O

 


 

SECTION: Step 1 modification of full vial of RoCKseq beads: preparation of reagents
9. Preparation of T4 polymerase mix: Prepare four 1.5 mL DNA LoBind tubes. Pipette into each tube: 260 µL T4 polymerase buffer, 130 µL 10 mM dNTPs, 857 µLddH2O and place on ice
SECTION: Step 1 modification of full vial of RoCKseq beads: preparation of reagents
10. Preparation of splint mix: Pipette 15 µL of 100 µM polyA oligo and 15 µL of 100 µM splint into new 1.5 mL DNA LoBind tube. If a mix of splints is used, pipette 15 µL of 100 µM polyA oligo and 15 µLof mix of splints (see Step 3)

 

SECTION: Step 1 modification of full vial of RoCKseq beads: preparation of reagents
11. Incubate splint mix in thermomixer at 75 °C for 5 min without shaking and place on ice
SECTION: Step 1 modification of full vial of RoCKseq beads: preparation of reagents
12. Preparation of beads: Resuspend the beads by gently pipetting up and down with a 1 mL pipette set to 500 µL being careful not to lose any supernatant. Immediately transfer the2 mL of barcoded beads provided by the manufacturer by pipetting 500 µL of BD Rhapsody barcoded beads into four new 1.5 mL DNA LoBind tubes and place on ice. After the transfer to each tube resuspend the remaining beads by pipetting up and down to allow for a similar amount of beads being transferred per replicate

 

SECTION: Step 1 modification of full vial of RoCKseq beads: preparation of reagents
13. Proceed immediately to “Washing BD Rhapsody beads”
SECTION: Step 2 modification of full vial of RoCKseq beads: washing BD Rhapsody beads
14. Place the four 1.5 mL DNA LoBind tubes containing the beads on a 1.5 mL magnetic stand
SECTION: Step 2 modification of full vial of RoCKseq beads: washing BD Rhapsody beads
15. Wait until liquid in tubes is clear, takes about 1 min to complete
SECTION: Step 2 modification of full vial of RoCKseq beads: washing BD Rhapsody beads
16. Gently remove supernatant with 1 mL pipette without disturbing the beads - the LoBind tube remains on the magnetic stand

 

SECTION: Step 2 modification of full vial of RoCKseq beads: washing BD Rhapsody beads
17. Remove first tube from magnetic stand and resuspend beads in at least600 µL Water buffer, gently pipette up and down at least 5 times to resuspend beads and place the tube on ice

 

SECTION: Step 2 modification of full vial of RoCKseq beads: washing BD Rhapsody beads
18.    Repeat Step 17 with the other three tubes
SECTION: Step 3 modification of full vial of RoCKseq beads: T4 polymerase elongation
22. Place the four 1.5 mL tubes with washed BD Rhapsody beads on 1.5 mL magnetic stand and wait until liquid is clear, takes about 1 min to complete

 

SECTION: Step 2 modification of full vial of RoCKseq beads: washing BD Rhapsody beads
19. Place the four 1.5 mL with washed BD Rhapsody beads on 1.5 mL magnetic stand
SECTION: Step 2 modification of full vial of RoCKseq beads: washing BD Rhapsody beads
20.   Repeat from Step 14 with TE/TW buffer processing one tube at the time as before
SECTION: Step 2 modification of full vial of RoCKseq beads: washing BD Rhapsody beads
21. Resuspend the beads in at least600 µLTE/TW buffer and place on ice

SECTION: Step 3 modification of full vial of RoCKseq beads: T4 polymerase elongation
23. Remove supernatant from first tube - the tube remains on the magnetic stand
SECTION: Step 3 modification of full vial of RoCKseq beads: T4 polymerase elongation
24. Resuspend beads from first tube with T4 polymerase mix (from Step 4) by gently pipetting up and down at least 5 times
SECTION: Step 3 modification of full vial of RoCKseq beads: T4 polymerase elongation
25. Place the tube on a rack (non magnetic) at Room temperature

 

SECTION: Step 3 modification of full vial of RoCKseq beads: T4 polymerase elongation
26.   Repeat Steps 23-25 with the remaining three tubes 

SECTION: Step 3 modification of full vial of RoCKseq beads: T4 polymerase elongation
27. Mix splint (from Steps 10-11) by pipetting with a 200 µL pipette set to 30 µl
SECTION: Step 3 modification of full vial of RoCKseq beads: T4 polymerase elongation
28. To each of the four tubes with beads containing the T4 polymerase mix add 6.3 µLof splint, using a new pipette tip each time
SECTION: Step 3 modification of full vial of RoCKseq beads: T4 polymerase elongation
29. Place the tubes with resuspended beads into the thermomixer at 37 °C and shake for 5 min at 

SECTION: Step 3 modification of full vial of RoCKseq beads: T4 polymerase elongation
30. After the incubation at Step 29, place the tubes on a (non-magnetic) rack at Room temperature
SECTION: Step 3 modification of full vial of RoCKseq beads: T4 polymerase elongation
31. Add 6.3 µLT4 polymerase to each of the four tubes
 


SECTION: Step 3 modification of full vial of RoCKseq beads: T4 polymerase elongation
32. Place the tubes on a MacsMix tube rotator for 10 minand rotate on second speed setting (at  )
 

SECTION: Step 3 modification of full vial of RoCKseq beads: T4 polymerase elongation
33. Transfer the tubes to a thermomixer at 75 °C for 10 min without shaking 
 

SECTION: Step 3 modification of full vial of RoCKseq beads: T4 polymerase elongation
34. During the 10 minutes incubation time in Step 33, prepare lambda exonuclease mix: in four 1.5 mL DNA LoBind tubes, pipette 95 µL lambda exonuclease buffer, 832 µL water in each tube and place on ice. Once the incubation at Step 33 is finished, place the tubes on ice for 1 min

SECTION: Step 3 modification of full vial of RoCKseq beads: T4 polymerase elongation
35. Wash BD Rhapsody beads as described above in the section Washing BD Rhapsody Beads (  ), after which resuspend in at least 200 µL TE/TW buffer and place on ice

SECTION: Step 4 modification of full vial of RoCKseq beads: lambda exonuclease digest
36. Place the four tubes containing the beads on a 1.5 mL magnetic stand, wait for 1 min and remove the supernatant, not disturbing the beads. The tubes remain on the stand.

SECTION: Step 4 modification of full vial of RoCKseq beads: lambda exonuclease digest
37. Remove the first tube from the stand and resuspend the beads using the lambda exonuclease mix (927 µL, from Step 34)

SECTION: Step 4 modification of full vial of RoCKseq beads: lambda exonuclease digest
38. Place the tube on a non-magnetic rack at Room temperature
 

SECTION: Step 4 modification of full vial of RoCKseq beads: lambda exonuclease digest
39.   Repeat Steps 37-38 with other three tubes
SECTION: Step 4 modification of full vial of RoCKseq beads: lambda exonuclease digest
40. To each of the four tubes with beads resuspended in lambda exonuclease mix add 21 µLof lambda exonuclease 
 


SECTION: Step 4 modification of full vial of RoCKseq beads: lambda exonuclease digest
41. Transfer the four tubes to a thermomixer at37 °Cfor 30 min without shaking 
SECTION: Step 4 modification of full vial of RoCKseq beads: lambda exonuclease digest
42. Transfer the tubes to a thermomixer set to 75 °Cfor 10 min without shaking
 

SECTION: Step 4 modification of full vial of RoCKseq beads: lambda exonuclease digest
43. After the incubation at Step 42, immediately place the tubes on ice for 1 min
SECTION: Step 4 modification of full vial of RoCKseq beads: lambda exonuclease digest
44. Wash BD Rhapsody beads as described above in the section Washing BD Rhapsody Beads (  ), after which resuspend in at least 200 µL TE/TW buffer and place on ice
SECTION: Step 5 modification of full vial of RoCKseq beads: final resuspension beads and storage
45. Place the four tubes containing the BD Rhapsody beads on the 1.5 mL magnetic stand and wait for1 min
SECTION: Step 5 modification of full vial of RoCKseq beads: final resuspension beads and storage
46. Remove supernatant from first tube and resuspend the beads in 250 µL TE/TW buffer by gently pipetting up and down at least 5 times and place on ice
SECTION: Step 5 modification of full vial of RoCKseq beads: final resuspension beads and storage
47.   Repeat step 46 with the other three tubes 
SECTION: Step 5 modification of full vial of RoCKseq beads: final resuspension beads and storage
48. Pool the resuspended beads into a new 1.5 mL Lobind tube
SECTION: Step 5 modification of full vial of RoCKseq beads: final resuspension beads and storage
49. Store RoCKseq modified beads at 4 °C . Beads are stable over time in TE/TW buffer. 
 

 

SECTION: Design of capture sequences
1. Before proceeding with the bead modification step, splints and fluorescent oligo need be designed and ordered
SECTION: Design of capture sequences
2. Points to keep into consideration when designing splints: 

The GC content of splints should be in the range of 40-60%. Higher GC content may impair reverse transcription (i.e. first strand synthesis). Also consecutive GC stretches of more than 4 bases should be avoided. Similarly a low GC content and longer stretches of A should be avoided in order to prevent dT-based capture of the target transcript

GC content upstream of splint: if the GC content of the transcript of interest upstream of the splint is too high (more than 5 consecutive G or Cs), this may impair reverse transcription (i.e. first strand synthesis)

Length of the splint: 24 nucleotides 

Place the capture whenever possible into the CDS of the transcript of interest: the 3’- and 5’ UTRs are less conserved and thus more prone to accumulate nucleotide polymorphisms that will hamper targeted capture. For long non-coding RNAs we suggest capturing the transcript in a conserved region whenever possible. Sequencing the locus in the strain used is recommended.

Vicinity to ROIseq primer: when performing RoCK and ROI, the splint should be chosen not more than 300 - 400 bp downstream of the ROIseq primer. This accounts for the sequence on the bead (primer, barcode, UMI, TSO). Please note adaptors for sequencing add to the final product size as well. 

G or a C at the 5’ end of the splint (and thus 3’ end of the capture) favor reverse transcription. 

The capture should not be overlapping with known splice junctions: this may be an issue if unknown splice variants are present (i.e. intron retention) 

SECTION: Design of fluorescent oligos
4. To design the fluorescent oligos, take the first 20 nucleotides from the 5’ end of the splint. 

The fluorescent oligos should be ordered in HPLC grade and in 0.2 µmol scale with a 5’ Atto647N modification and diluted in ddH2O to generate a 100 µM stock solution.

 

SECTION: Design of capture sequences
3. Splint sequences

IMPORTANT: all splints are 5' phosphorylated

The sequence of the splint for the modification of TSO oligos on BD Rhapsody “Enhanced Cell Capture Beads V2” is as follows: 

5’ -24 or 25 nt coding sequence followed by a constant sequence-3’: 

5’-NNNNNNNNNNNNNNNNNNNNNNNNCATACCTACTACGCATA-3’ 

where the CATACCTACTACGCATA is the reverse complement of the TSO sequence on the beads. 

The polyA protective oligo used on the barcoded beads is 18 nucleotides in length: 

5’-AAAAAAAAAAAAAAAAAA-3’

The oligos should be ordered in 0.2 µmol scale, HPLC grade, with 5’ phosphorylation. Before use, resuspend the oligos in ddH2O to generate a 100 µM stock solution.

IMPORTANT: 

To modify RoCKseq beads with multiple capture sequences, mix the splints in the desired ratio. For example, to modify RoCKseq beads with the same amount of three splints (33% each), pipette5 µLof each splint and mix with 15 µL of 100 µM polyA oligo

The modification of RoCKseq beads can be titrated to achieve different amounts of modification on TSO oligos. The titration is achieved by mixing the splint(s) with the protective TSO oligo. This oligo is also 5' phosphorylated. For example, to achieve a 50% of RoCKseq modification, a mix of 7.5 µL of splint(s) and 7.5 µL of protective TSO oligo is generated and mixed with 15 µLof 100 µM polyA oligo

SECTION: Fluorescent assay for the detection of RoCKseq modification and integrity of DNA oligos on beads
52. Prepare TE/TW and Water buffers in 50 mL Falcons and place on ice
 

SECTION: Fluorescent assay for the detection of RoCKseq modification and integrity of DNA oligos on beads
53. Thaw fluorescent oligos (100 µM) at Room temperature

 

SECTION: Fluorescent assay for the detection of RoCKseq modification and integrity of DNA oligos on beads
54. Prepare lysis buffer in a 1.5 mL Eppendorf tube and place on ice: per sample add 1 µL1 M DTT (part number 650000063, BD RhapsodyTM Enhanced Cartridge Reagent Kit) to 188 µL µl BD Rhapsody lysis buffer (part number 650000064, BD RhapsodyTM Enhanced Cartridge Reagent Kit) and mix thoroughly
SECTION: Fluorescent assay for the detection of RoCKseq modification and integrity of DNA oligos on beads
51. Preheat a thermomixer to 46 °C
SECTION: Fluorescent assay for the detection of RoCKseq modification and integrity of DNA oligos on beads
55. Dilute 10 µLof fluorescent oligo (100 µM) 1:10 in ddH2O and place on ice. Keep in the dark
 


SECTION: Fluorescent assay for the detection of RoCKseq modification and integrity of DNA oligos on beads
56. Place previously modified BD Rhapsody barcoded beads on ice
SECTION: Fluorescent assay for the detection of RoCKseq modification and integrity of DNA oligos on beads
58. Pipette 20 µLof RoCKseq modified beads per condition in a new Eppendorf tube and place on ice 
SECTION: Fluorescent assay for the detection of RoCKseq modification and integrity of DNA oligos on beads
60. Add 188 µL lysis buffer + DTT (from Step 54) per condition and place the tube on a non-magnetic rack at room temperature
 

SECTION: Fluorescent assay for the detection of RoCKseq modification and integrity of DNA oligos on beads
61. Add 8 µLof the 10 µM fluorescent oligo per sample (prepared at Step 55) and gently pipette up and down to mix
SECTION: Fluorescent assay for the detection of RoCKseq modification and integrity of DNA oligos on beads
62. Incubate samples for 30 min at 46 °C shaking at   in the dark (for example covering the thermomixer block with aluminum foil)
SECTION: Fluorescent assay for the detection of RoCKseq modification and integrity of DNA oligos on beads
63. Wash beads as described in Step 2 modification of full vial of RoCKseq beads: washing BD Rhapsody beads (  ) and resuspend in 300 µL TE/ TW buffer
SECTION: Fluorescent assay for the detection of RoCKseq modification and integrity of DNA oligos on beads
64. Strain beads in a Falcon 5 mL Round Bottom Polystyrene Test Tube, place on ice, keep in dark and measure fluorescent intensity at a FACS analyser. 

Vortex beads before loading the sample. If the event rate drops, stop acquisition and vortex beads again. 

Measure 1000 events per sample.

 

 

 

SECTION: RoCKseq bead modification protocol for splint testing
5. IMPORTANT: the protocol described below refers to the modification of a full vial of BD Rhapsody barcoded beads. Alternatively, to test the efficacy of the bead modification with new capture sequences, the protocol can be adapted to modify a small aliquot of beads. 

Instead of 2 mLof BD Rhapsody barcoded beads per sample, 20 µLof beads can be used. The same protocol can be used with the following changes:

Step 9: T4 polymerase mix: 40 µL buffer, 20 µL10 mM dNTPs, 136 µL ddH2O
Step 28: 1 µL of polyA – splint mix 
Step 31: 1 µLT4 polymerase enzyme
Step 34: lambda exonuclease mix: 15 µL reaction buffer, 132 µL ddH2O 
Step 40: 3 µL lambda exonuclease enzyme

The fluorescent assay protocol can be used as described below, with all 20 µL of modified beads being used as input. 
SECTION: Fluorescent assay for the detection of RoCKseq modification and integrity of DNA oligos on beads
57. Wash unmodified beads used as controls as described in Step 2 modification of full vial of RoCKseq beads: washing BD Rhapsody beads (  ) and place on ice
SECTION: Fluorescent assay for the detection of RoCKseq modification and integrity of DNA oligos on beads
59. Place the tubes containing the 20 µL of beads on a 1.5 mL magnetic stand, wait for 1 min and remove the supernatant, not disturbing the beads. The tubes remain on the stand.
SECTION: Fluorescent assay for the detection of RoCKseq modification and integrity of DNA oligos on beads
65. 
 

SECTION: Fluorescent assay for the detection of RoCKseq modification and integrity of DNA oligos on beads
50. Recommended conditions for fluorecent assay

 

 

SECTION: Design of ROIseq primers
66. ROIseq primers should be designed directly 5’ (max. 10bp upstream) to the region of interest (ROI). The length of the primers is 12 nucleotides. Since 12 nucleotides will be included in the cDNA sequencing read (HTS), the ROIseq primer must be in close proximity to the ROI. 

Depending on the ROI to be detected, it may be advantageous to position the ROIseq primer further upstream to the sequence of interest. This is the case for example for fusion transcripts, in which having a longer stretch to map on both sides of the fusion breakpoint is beneficial. In this case we recommend using longer read length and placing the ROIseq primer 20-30 bp upstream to the ROI itself. 

The ROIseq primer has the following structure: 

5’-TCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNN-3’, the N being the sequence of the ROIseq primer which identical to the coding strand.

An additional consideration when designing ROIseq primers is that ideally the read generated after ROIseq priming should be unique, i.e. doesn’t map to multiple loci. 

ROIseq primers should be ordered in HPLC grade and at 0.2 µmol scale and resuspended in DNA Supension buffer from Teknova (T0221). 
SECTION: RoCK and ROI library generation
67. RoCK and ROI library generation follows the standard BD Rhapsody workflow (mRNA capture, reverse transcription and exonuclease treatment: Doc ID: 210966; library generation Doc ID: 23-21711-00) with the following adaptations (steps 67.1-67.4 indicate the steps in the standard protocols where the changes occur): 
SECTION: RoCK and ROI library generation
67.1. Resuspending barcoded beads prior to loading on cartridge: to account for the bead loss during modification, resuspend the RoCKseq beads in 680 µLSample Buffer (Cat. No. 650000062, BD RhapsodyTM Enhanced Cartridge Reagent Kit) instead of 750 µLprior to loading on the BD Rhapsody cartridge
SECTION: RoCK and ROI library generation
67.2. Random priming and extension: if a single ROIseq primer is added, dilute 1 µL of the 100 µM primer 1:10 in ddH2O and pipette 4 µLof the diluted mix during the Random Priming and Extension step (after pipetting the 174 µL ). Add the ROIseq primers after the beads are resuspended in the Random Primer mix. 

If multiple ROIseq primers are used, mix 1 µL of each ROIseq primer (100 µM), add ddH2O up to10 µLand add 4 µL to the mix. 
SECTION: RoCK and ROI library generation
67.3. RPE PCR: add 1 µL of 100 µM T primer to each sample after the RPE PCR mix is added to the Purified RPE product. 
 

SECTION: RoCK and ROI library generation
67.4. Indexing PCR: for indexing of RoCKseq libraries, a separate PCR is performed substituting 5 µL of the Library Forward Primer (BD RhapsodyTM Enhanced Cartridge Reagent Kit, part number 91-1085) with 5 µL of 100 µM of a custom indexing primer. The same primary library and reverse primers are used as recommended by the manufacturer. The reaction is thus as follows: 

For WTA library (from BD Rhapsody Doc ID: 23-21711-00): 

 

 For TSO library: 
 
 
 
IMPORTANT: RoCKseq and dT-based libraries of a given sample should be indexed with the SAME BD Rhapsody Library Reverse Primer and will thus have the same 8 bp index. The two data modalities are then separated bioinformatically (see Step 69) 
 

SECTION: RoCK and ROI library generation
67.5. If no ROIseq is being performed omit step 67.2 
SECTION: Sequencing
68.1. We recommend pooling the WTA and TSO libraries in a 1:1 ratio. 

For sequencing of pooled libraries including at least one RoCKseq modified sample (with or without ROIseq primers), a custom R1 primer should be spiked in (see Materials).

The length of R1 should be 60 bp, while the length of R2 may vary depending on the ROI of interest (see section Design of ROIseq primers, Step 66). We recommend using an R2 of 62 bp for ROIs such as point mutations and splice junctions and an R2 of 150 bp for fusion breakpoints and CRISPR target sites. 
SECTION: RoCK and ROI library generation
68. 
 


 


 


SECTION: Data analysis
69. RoCK and ROI data can be analysed using our custom pipeline, found at https://zenodo.org/records/11070201 under the GPLv3 terms. For downstream data processing please see https://zenodo.org/records/11124929.