Nebnext ultra 2 dna library prep with sample purification beads kit

Cells were subjected to Drop-Seq following the original protocol² with the following modifications. Barcoded beads were resuspended in the yeastDrop-Seq solution containing the reagents as shown in Table 1. Cells were diluted to 50 cells/μL. After initial droplet formation, droplets were incubated for 30 min at 30 °C to ensure Zymolyase breakdown of cell walls followed by cell bursting (Supplementary Fig. 1). After incubation, droplet quality was evaluated and >95% droplet-uniformity remained intact.Table 1

yeastDrop-Seq solution.

Solution (total volume)	Reagent	Reagent volume
yeastDrop-seq solution (2223.5 μL)	Zymolyase (Zymo Research E1004), 2 Units/μL	37.5 μL
	Lysis buffer (no DTT)	1.5 mL
	1 M DTT	75 μL
	Zymolyase buffer	600 μL
	10% SDS (final concentration at 0.05%)	11 μL
Lysis buffer (no DTT)2 (950 μL)	H2O	500 μL
	20% Ficoll PM-100	300 μL
	Sarkosyl	10 μL
	0.5 M EDTA	40 μL
	2 M Tris pH 7.5	100 μL
Zymolyase buffer37 (CSH protocols Zymolyase Buffer) (100 mL)	2 M Sorbitol	50 mL
	1 M K2HPO4	4.2 mL
	1 M KH2PO4	0.8 mL
	EDTA (0.5 M, pH 8)	1 mL
	H2O	44 mL

Downstream breakage of oil droplets followed by reverse transcription, exonuclease I treatment, and PCR for cDNA amplification were carried out as per the Drop-Seq protocol² . cDNA was then tagmented using the NEBNext Ultra II DNA Library Prep with Sample Purification Beads kit (NEB #E7103S).
Sequencing was done using the Illumina HiSeq2500 platform with 2 × 100 read pairs.