High RNA quality extracted from the tolerant crop Cyamopsis tetragonoloba (L.) despite possession of low RNA integrity number

Figures & data

Table 1. Extraction method, library construction and sequencing information.

RNA samples	Label	Extraction method	Library construction type (insert size)	Sequencing platform	Read length and sequencing mode	Amount of data
Control (C)	Ph-C	Chloroform extraction	Truseq stranded mRNA library (350)	Illumina Hiseq2500	101bp PE	3Gb per sample Raw data only
	Q-C	RNeasy® Plant Mini
Salinity treated (T)	Ph-T	Chloroform extraction
	Q -T	RNeasy® Plant Mini

Figure 1. Separation of DNase treated total RNA using the Phenol based method (Lanes 2-7 both control and treated samples) and RNeasy kit (Lanes 8-13, both control and treated samples), on 1% formaldehyde (w/v) denaturing 1.2% agarose gel electrophoresis showing multiple ribosomal RNA bands with no signs of RNA degradation. [M, 100 bp DNA Ladder].

Table 2. Purity or integrity and average yields of total RNA extracted by the two methods and determined spectrophotometrically (nanodrop vs Bioanalyzer vs Tapestation).

Sample name*	Nanodrop			Bioanalyzer		Tapestation
	Total amount (μg / 50 μL)	A260/A280	A260/A230	Total amount (μg / 50 μL)	RIN	RIN^e
Phenol-Control	18.37	1.91	0.40	22.13	5.7 ^a	5.6 ^a
	±3.59	±0.06	±0.03	±1.4	±0.06	±0.25
Phenol-Treated	16.70	1.89	0.57	18.21	5.7 ^a	5.7 ^a
	±1.83	±0.09	±0.09	±0.85	±0.21	±0.15
Qiagen-Control	8.74	2.07	2.45	10.59	6.3 ^b	6.4 ^b
	±1.26	±0.07	±0.08	±0.31	±0.10	±0.21
Qiagen-Treated	7.75	2.02	2.56	8.44	6.1 ^b	6.4 ^b
	±0.78	±0.06	±0.10	±0.09	±0.10	±0.26

* Three replicates.

Means followed by a common letter are not significantly different by the Duncan-test at the 95% level of significance.

Figure 2. (a) "Gel Image" display of microcapillary electrophoresis electropherograms demonstrating the integrity of the RNA in the Agilent 2100 Bioanalyzer. The lower marker band across all samples is the electrophoresis marker (roughly 22 s). Phenol based method (Lanes 2-7, both control and treated samples) and RNeasy kit (Lanes 8-13 both control and treated samples); (b) injected ladder in RNA 6000 Nano total RNA kit.

Figure 3. Electropherogram of the RNA of the three biological replicates of the control samples (Qiagen RNeasy Kit) using 2100 Bioanalyzer RIN: 6.4.

Table 3. Plant ribosomal RNA.

Sedimentation coefficient	Ribosomal subunit
Cytosolic rRNA content
25S − 5.8S - 5S	larger subunit (60S)
18S	smaller subunit (40S)
Chloroplast rRNA content
23S - 5S − 4.5S	larger subunit (50S)
16S	smaller subunit (30S)
Mitochondrial rRNA content
24S - 5S	larger subunit (60S)
18.5S	smaller subunit (44S)

Figure 4. Electropherogram of post-library construction check from the Agilent 2200 TapeStation system for RNeasy extracted sample (Q-C1).

Table 4. mRNA-seq Library quality check Result - Agilent 2200 TapeStation.

Library Name *	Conc. (ng/μL)	Conc. (nmol/L)	Size (bp)
Phenol-Control	36.29	187.78	298
	±0.33	±0.99	±2.52
Phenol-Treated	38.00	195.59	299
	±8.24	±41.68	±1.53
Qiagen-Control	39.70	207.05	298
	±3.72	±18.47	±2.89
Qiagen-Treated	37.66	198.89	299
	±7.08	±36.30	±1.00

* Three replicates.

Table 5. Basic statistics of the retrieved raw data.

Sample ID	Total read bases (bp)	Read count	GC content	Q20 %	Q30 %
Ph-C1	3,929,626,392	38,907,192	43.70	97.49	95.83
Ph-C2	3,993,488,490	39,539,490	43.90	96.91	95.02
Ph-C3	3,926,584,070	38,877,070	44.02	97.41	95.71
Ph-T1	3,465,423,322	34,311,122	44.56	96.84	94.97
Ph-T2	3,554,211,210	35,190,210	44.44	98.23	96.57
Ph-T3	3,488,268,916	34,537,316	44.64	97.65	95.76
Q-C1	3,820,792,024	37,829,624	44.76	98.15	96.45
Q-C2	3,874,987,412	38,366,212	45.30	97.58	95.72
Q-C3	3,728,755,370	36,918,370	44.62	98.41	96.75
Q-T1	3,836,036,560	37,980,560	44.82	97.83	95.94
Q-T2	3,788,794,618	37,512,818	44.94	98.33	96.63
Q-T3	3,765,113,754	37,278,354	45.48	97.76	95.90

Q20% - Q30%: percentage of reads with a Phred quality score of over 20 and 30, respectively.

Figure 5. Quality score across all bases [RNeasy extracted sample (Q-C1)] - dataset ready for assembly - Forward R1 (a) and Reverse R2 (b).

Table 6. RNA-seq Paired-end data quality and adapter trimming using Trimmomatic.

Sample	Input reads	Both surviving	%	Sample	Input reads	Both surviving	%
Ph-C1	38,907,192	36,245,940	93.16%	Q-C1	37,829,624	35,658,204	94.26%
Ph-C2	39,539,490	35,795,100	90.53%	Q-C2	38,366,212	35,154,960	91.63%
Ph-C3	38,877,070	35,980,728	92.55%	Q-C3	36,918,370	34,574,054	93.65%
Ph-T1	34,311,122	31,861,308	92.86%	Q-T1	37,980,560	35,686,534	93.96%
Ph-T2	35,190,210	32,688,186	92.89%	Q-T2	37,512,818	35,258,298	93.99%
Ph-T3	34,537,316	31,808,868	92.10%	Q-T3	37,278,354	34,743,426	93.20%
Total	221,362,400	204,380,131	92.33%	Total	225,885,938	211,075,475	93.44%

Table 7. De-novo transcriptome assembly, general statistics.

Sample	Transcriptome size	Transcripts	N50	≥ 1000 bp scaffolds (transcripts)
Phenol based RNA	∼281 Mbp	65,366	3153	49,463
RNeasy based RNA	∼295 Mbp	68,148	3354	51,381

Data availability

The authors confirm that the data supporting the findings of this study are available within the article.