Table 1.

Protocol modifications

	Manufacturer’s protocol	Miniaturized protocol
Poly(A) isolation (1/20th)	Single mRNA elution and re-bound for 5 min to poly(A) beads.	Two rounds of mRNA elution and rebinding. Incubation time was increased from 5 to 10 min.
		mRNA eluted from beads in 11.5 µl of First Strand Synthesis Reaction Buffer and Random Primer.	mRNA was eluted in 1.5 µl First Strand Synthesis Reaction Buffer and Random Primer mix instead of 1.15 µl.a
Ribosomal depletion (1/6th)	Ribosomal depleted RNA eluted in 7 µl of nuclease-free water after clean.	Increased elution volume of the ribosomal depleted RNA from 1.17 to 1.5 µl.
		5 µl of the First Strand Synthesis Reaction Buffer and Random Primer mix added to 5 µl of rRNA depleted sample for fragmentation.	750 nl of the First Strand Synthesis Reaction Buffer and Random Primer is added to 750 nl of rRNA depleted sample.a
cDNA synthesis (1/10th)	Fragment RNA by incubating at 94°C for 15 min.	Reduced the incubation time for fragmentation to 10 min at 94°C.
		During first-strand synthesis, samples are incubated at 42°C for 15 min.	Increased time of incubation at 42°C to 50 min.
Library preparation (1/10th)	For 100 ng of total RNA input, stock NEBNext Adaptor is diluted 25-fold for adaptor ligation.	For 100 ng of total RNA input, stock NEBNext Adaptor is diluted 20-fold for adaptor ligation.	2.5 µl of the diluted adaptor used for ligation reaction.	Increased volume of diluted adaptor added from 250 to 500 nl.
		Adaptor-ligated DNA is eluted in 17 µl of 0.1× Tris-EDTA (TE) buffer after bead clean.	Increased elution volume for adaptor-ligated DNA from 1.7 to 2 µl.
		Final libraries purified by adding 45 µl (0.9× cut) of sample purification beads.	Final libraries are purified by a two-part SPRI clean. First with 6 µl (1.2×) SPRI beads followed by addition of SPRI buffer equivalent to a 0.9× cut.

Protocol steps for full-volume reactions with corresponding description of changes made. All reactions were miniaturized proportionally except at steps noted here.

^aTransition step to cDNA synthesis, which is performed at a 1/10th reaction scale.