TITLE: Hybrid selection protocol using 10x Single-Cell RNA-Seq assay library
AUTHORS: Xian Adiconis, Joshua Z Levin

[DESCRIPTION]
This protocol is for target enrichment of cDNA libraries generated with 10x Genomics single-cell RNA-seq assays. The protocol consists of parts of vendor provided protocols with minor modification. The hybridization and PCR part is based on "CG000059_DemonstratedProtocolExome_RevC" (10x Genomics) and the capture part is based on "SureSelectXT Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library Protocol Version C3, September 2019" (Agilent).

[STEPS]
SECTION: Sample preparation
1. Use 750 ng of cDNA library generated with the 10x Chromium single-cell RNA-seq assay.

SECTION: Sample preparation
2. Add 1 µL each of TS-p5 and TS-p7 blocking oligos.


SECTION: Sample preparation
3. If the total volume exceeds 3.4 µL, use a speed-vac and set the temperature at 30 °C to reduce the volume to the desired volume of 3.4 µL.

SECTION: Buffer and reaction mix preparation
4. Prepare the Hybridization Buffer with reagents from the SureSelectXT kit at Room temperature 
Prepare the volume for at least 5 reactions to ensure accurate pipetting.
 
 
 
SECTION: Buffer and reaction mix preparation
5. Prepare the block mix at 4 °C 
 
 
 
SECTION: Sample and block denaturing
6. Add 5.6 µLBlock mix to the concentrated 3.4 µLsample, mix well and transfer to the 0.2ml PCR tube strip (Axygen), and place into a thermocycler following
Thermocycler Conditions: (Lid@105 °C, 100 µl)
95 °C, 5 min  
65 °C,    

SECTION: Hybridization
7. While step 6 mix is incubated at 65 °C for 5 min, prepare the following at Room temperature
 
 
 Mix well by high speed vortexing for 5 s, spin down briefly, add into the sample and block  mix at 65 °C, mix by pipetting 8-10x, use a new cap strip to seal the tubes
Incubate at 65 °C for 960 min to 1440 min

SECTION: Library capture
8. In a strip tube, use 50 µLMyOne Streptavidin T1 beads per sample, wash with 200 µL SureSelect Binding buffer 3 times, resuspend in 200 µLSureSelect Binding buffer.


SECTION: Library capture
9. Bring the washed beads from step 8 near the thermocycler, add the 65 °Creaction mix (~27 µL left) right into the 200 µLbeads, gentle pipetting mix, incubate at Room temperature for 30 min, pipetting mix 6 times every 5 min

SECTION: Library capture
10. Place on a magnet stand to pellet the beads and remove the supernatant once the solution appears clear. Resuspend the beads in 200 µLSureSelect Wash buffer 1, incubate at Room temperaturefor 15 min

SECTION: Library capture
11. Meanwhile, pre-warm SureSelect Wash buffer 2 at 65 °C in strip tubes with 200 µL per well and 3 wells per reaction on a 96-well heating block (or a thermocycler with reaction volume capacity of 200 µL)

SECTION: Library capture
12. Place step 10 reaction mix on a magnet stand to pellet the beads and remove the supernatant once the solution appears clear. 
SECTION: Library capture
13. Resuspend the beads in 200 µLpre-warmed SureSelect Wash buffer 2, incubate at 65 °Cfor 10 min Place the reaction mix on a magnet stand to pellet the beads and remove the supernatant once the solution appears clear.
SECTION: Library capture
14. Repeat step 13 two more times and total three washes with pre-warmed SureSelect Wash buffer 2. And make sure all the wash buffer has been removed in the final wash.

SECTION: Library capture
15. Resuspend the beads in 30 µL H2O and keep on ice.

SECTION: Enrichment PCR
16. On ice, assemble the following mix
 
 Thermocycler Conditions: (Lid@105 °C, 100 µl)
 
 98 °C ,45 s 
then 9 cycles of
98 °C , 15 s 
60 °C , 30 s 
72 °C , 30 s 
then
72 °C , 1 min 
4 °C ,   

SECTION: Enrichment PCR
17. Place the PCR reaction tube on a magnet stand, wait until it clears and recover 100 µLsupernatant to a new PCR tube strip. 

SECTION: Enrichment PCR
18. Add 180 µL SPRI beads (1.8x) to the recovered PCR product, mix well by pipetting, pellet the beads on a magnet stand, after supernatant removal, wash with 300 µL80% ethanol twice, elute with 20 µLbuffer EB (10 mM Tris-HCl, pH 8.5).

SECTION: Enrichment PCR
19. QC with Quant-it (Thermo Fisher Scientific) and BioAnalyzer DNA HS assay (Agilent).