Materials and Reagents

RNeasy Micro Kit (QIAGEN, catalog number: 74004 )
WT-Ovation Pico RNA Amplification System (Nugen, catalog number: 3300 )
WT-Ovation Exon Module (Nugen, catalog number: 2000 )
Qubit dsDNA HS Assay Kit (Life Technologies, Invitrogen™, catalog number: Q32851 )
Tris-EDTA pH 8.0, molecular biology grade (Life Technologies, Ambion^®, catalog number: AM9849 )
TruSeq DNA Sample Preparation Kit (Illumina, catalog number: FC-121-2001 )
MinElute PCR Purification Kit (QIAGEN, catalog number: 28004 )
QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704 )
Certified Low Range Ultra Agarose (Bio-Rad Laboratories, catalog number: 161-3107 )

Equipment

BD FACSAria Fluorescence activated cell sorter
NanoDrop 1000
PCR Thermal Cycler
Sonic Dismembrator 550 (Thermo Fisher Scientificc)
Microtip (Misonix Inc, catalog number: 419 )
Fluorometer
Agarose gel running apparatus and UV-light transilluminator
Illumina HiSeq 2000

Figure 1. Overview of the protocol and main reagents used

Procedure

Sort 4,000 cells directly in 75 μl of RLT buffer (Qiagen RNeasy Micro Kit) using BD FACSAria cell sorter.
Isolate total RNA from sorted cells using the RNeasy Micro Kit according to manufacturer’s protocol without the addition of β-mercaptoethanol to RLT buffer. Perform on column DNA digestion for 15 min according the manufacturer’s instructions using the DNase that is provided with the RNeasy Micro Kit and elute in 14 μl of RNase/DNase free water. Yield of total RNA using a NanoDrop 1000 ranges between 30-80 ng.
Amplify RNA and generate cDNA with complementary sequences to the original mRNA using the WT-Ovation Pico RNA Amplification System according to manufacturer’s instruction. Expected yield of amplified cDNA measured on a NanoDrop 1000 is between 5-10 μg. A quality control step can be included at this point by performing Real-Time PCR for known transcripts that are expected to be present in the amplified cDNA pool.
Generate sense-strand cDNA targets from 3 μg of complementary sequence cDNA using the WT-Ovation Exon Module kit according to manufacturer’s instructions.
Sonicate 3 μg of sense-strand cDNA that is diluted to a final volume of 400 μl Tris-EDTA pH 8.0 buffer to generate cDNA fragments in the 200-500 bp range. Sonication with Sonic Dismembrator 550 and a 419 Microtip is performed on ice with six pulses of 20 sec each (magnitude setting of 3) and a 60 sec rest interval.
Quantify sense-strand sonicated cDNA on a fluorometer using the Qubit HS dsRNA assay kit according to manufacturers’ instructions and aliquot 30 ng for generating the Sequencing library.

Perform End Repair (Reagents are part of the TruSeq DNA Sample Preparation Kit)

Sense-strand cDNA (30 ng)	30 μl
Water	10 μl
T4 DNA Ligase Buffer with 10 mM ATP	5 μl
10 mM dNTP mix	2 μl
T4 DNA Polymerase (3 U μl^-1)	1 μl
Klenow DNA Polymerase (1 U μl^-1 diluted with water)	1 μl
T4 PNK (10 U μl^-1)	1 μl

20 °C 30 min on a thermal cycler

Purify using the MinElute PCR Purification Kit according to manufacturer’s instructions and elute in 34 μl of RNase/DNase free water. Use all eluted cDNA in following step.
Adenylate 3’ ends (Reagents are part of the TruSeq DNA Sample Preparation Kit)
cDNA
34 μl
10x Klenow buffer
5 μl
1 mM dATP
10 μl
Exo-Klenow (5 U μl^-1)
1 μl
37 °C 30 min on a thermal cycler
Purify using the MinElute PCR Purification Kit according to manufacturer’s instructions. Elute with 10 μl of RNase/DNase free water.
Ligate paired-end adapters (reagents are part of the TruSeq DNA Sample Preparation Kit)
cDNA
10 μl
2x Ligase buffer
15 μl
Adapter oligo mix (1/10 dilution in water)
1 μl
DNA ligase
4 μl
Room temperature 15 min
Purify the ligation reaction using the MinElute PCR Purification Kit according to manufacturer’s instructions.
Size select the cDNA library by running the purified PCR product through a 1.5% Agarose Gel, cutting a smear in the range of 150-300 bp, purifying using the QIAquick Gel Extraction Kit according to manufacturer’s instructions and eluting with 36 μl of RNase/DNase free water. Note that DNA concentration is very low therefore there is no visible DNA band on the gel.
PCR amplification of the adapter modified DNA fragments (Reagents are part of the TruSeq DNA Sample Preparation Kit)
DNA
36 μl
5x Phusion buffer
10 μl
10 mM dNTPs
1.5 μl
PCR primer 1.1
1 μl
PCR primer 1.2
1 μl
Phusion DNA Polymerase (2 U μl^-1)
0.5 μl
Use the following protocol on a thermal cycler
1. 98 °C 30 sec
2. 18 cycles of
  98 °C 10 sec
  65 °C 30 sec
  72 °C 30 sec
3. 72 °C 5 min
4. 4 °C hold

Validate library by running the amplified DNA through a 1.5% Agarose Gel. The majority of the amplified cDNA should appear between the 150-300 bp range. Primer dimers and adapters appear at the 30-60 bp range.
Purify library by gel extracting the DNA that appears in the 150-300 bp range using the QIAquick Gel Extraction Kit according to manufacturer’s instructions and eluting with 15 μl RNase/DNase free water (Figure 2). Quantify library final yield on a fluorometer using the Qubit HS dsDNA assay according to manufacturer’s instructions. The libraries generated using this protocol yielded between 200-700 ng.

Figure 2. Example of gel extracted band alongside 100 bp ladder. Adapters visible around 30-60 bp.
A quality control step can be included at this point by performing Real-Time PCR for known transcripts that are expected to be present in the generated library. If a quality control step was performed at step 3, before generating the library, the results should be similar.

Run the cDNA library on an Illunina HiSeq 2000. Alignment statistics for all samples analyzed as well as correlation scores on the 3 replicates of hESC RNA-Seq libraries are shown in Tables 1 and 2.

Table 1. Alignment statistics

Sample	Total reads	Uniquely aligned reads (% total)
Testis 16 wk	116,399,186	35,497,900	30.5
Testis 16.5 wk	153,982,474	37,907,565	24.6
Ovary 16 wk	102,837,931	44,784,216	43.5
Ovary 16.5 wk	211,659,065	93,766,963	44.3
hESCs 1	161,037,803	39,339,764	24.4
hESCs 2	133,355,463	38,571,046	28.9
hESCs 3	104,997,235	37,164,448	35.4

Table 2. Correlation scores for hESC RNA-Seq libraries

	hESCs 1	hESCs 2	hESCs 3
hESCs 1	1	0.96	0.96
hESCs 2	0.97	1	0.97
hESCs 3	0.96	0.97	1

Acknowledgments

A.T.C. is supported by funds from the NIH/NICHD 2 R01 HD058047.

References

Gkountela, S., Li, Z., Vincent, J. J., Zhang, K. X., Chen, A., Pellegrini, M. and Clark, A. T. (2013). The ontogeny of cKIT+ human primordial germ cells proves to be a resource for human germ line reprogramming, imprint erasure and in vitro differentiation. Nat Cell Biol 15(1): 113-122.