Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

Table 2 Sample specifications and input details for SMART-seq V4 cDNA synthesis and amplification and for Nextera XT library prep

sample	RNA isolation kit	# cells	RNA Concentration	RQN	input SMART-seq v4	# amplification cycles	cDNA concentration	input cDNA Nextera XT
A_1	RNAqueous	20,000	0.75 ng/μl	9.6	9.5 μl	11	0.476 ng/μl	1 ng
Q_1	RNeasy	20,000	1.20 ng/μl	9.6	9.5 μl	11	1.24 ng/μl	1 ng
A_2a	RNAqueous	20,000	0.79 ng/μl	9.2	6 μl	13	13.6 ng/μl	1 ng
A_2b	RNAqueous	20,000	0.79 ng/μl	9.2	6 μl	13	15.6 ng/μl	1 ng
Q_2a	RNeasy	20,000	1.02 ng/μl	9.6	6 μl	13	16.1 ng/μl	1 ng
Q_2b	RNeasy	20,000	1.02 ng/μl	9.6	6 μl	13	16.2 ng/μl	1 ng
A_unsorted	RNAqueous	1 embryo	1.5 ng/μl	10	9.5 μl	11	3.06 ng/μl	1 ng
Q_unsorted	RNeasy	1 embryo	1.5 ng/μl	10	9.5 μl	11	5.95 ng/μl	1 ng

All samples, except A_unsorted and Q_unsorted, are 20,000 EGFP positive sorted cells from the same pool of Tg(fli1a:EGFP) zebrafish and RNA concentration was measured with the Fragment Analyzer. RNAqueous 2a and 2b and RNeasy 2a and 2b are technical replicates, starting from the same RNA. A_unsorted and Q_unsorted samples were obtained by isolating RNA from 1 embryo, RNA was measured with the Nanodrop (ThermoScientific) and RNA was diluted to 1.5 ng/μl to start with roughly the same input. cDNA concentration of all samples was measured with the Qubit dsDNA HS Assay kit (Invitrogen)

ISSN: 1471-2164