This method sequences both large coding/noncoding RNA, and small RNA from a single cell.

Single sperm Large and Small RNA seq:

 

Buffer Preparation:

 

 

Make 2X sperm lysis buffer:

 

160µl H20

100µl 2% SLS

40µl 1M Tris-HCL pH8

20µl 2M NaCl

80µl 1M DTT

400µl

 

Make Adenylated 3’ ligation adaptor:

 

**3’ adaptor adenylation should be done in bulk. Minimum 5 reactions.

 

1µl 100µM ligation adaptor (100pmol input)

2µl 10X buffer

2µl 1mM ATP

2µl Mth RNA ligase

13µl NF-H20

20µl

 

65˚C 1hr, 85˚C 5min, 4˚C hold

 

• pool 5 reactions together, purify with kappa beads with isopropanol alcohol

 

100µl pooled reaction

100µl Beads

270µl Isopropanol

 

Elute with 20µl NF-H20.

 

**optional: check the yield with Agilent Bioanalyzer DNA HS.

 

 

 

 

 

 

 

PartI:

 

Lysis:

 

~1µl sperm in PCR tube

1µl buffer 1 2X

2µl total

 

70˚C 5min, 4˚C hold

 

1.5µl H2O

0.22µl Tween-20 40%

Mix by pipetting, Room Temperature 5min

 

PolyA:

 

0.75µl First Strand Buffer 5X

0.25µl ATP (0.5mM)

0.25µl polyA polymerase

0.125µl RNaseIN

5.125µl

 

Mix by pipetting, place quickly on thermocycler

 

16˚C 5min, 65˚C 10min, 4˚C hold

 

Add: 1µl Barcode oligo in 96 well plate (0.025µM)

 

**Be very careful not to cross-contaminate the primers. Do not reuse plate film after taken the film off.

 

65˚C 5min, Snap cool on ice

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

* make 1miilion fold dilution of ERCC spike in #2

RT:

 

0.75µl FS buffer 5X

0.25µl DTT 100mM

0.5µl dNTP 10mM

0.25µl RNaseIn

0.5µl SSII

1µl ERCC Spikein #2 (1M dilution)

0.375µl Betain 5M

0.25µl H2O

 

10µl total

25˚C 10min, 42˚C 90min, 70˚C 10min, 4˚C hold

 

ExoI Digestion:

• pool 16 samples together, add 5µl ExoI to each Rxn. 37˚C 45min, 4˚C hold
• purify with 1.5X Kapa beads, elute with 12µl 10mM Tris
• Again, pool the 6 purified samples together, purify with 1X KAPA beads, elute with 12µl 10mM Tris. We now have 96 samples pooled together.

 

 

 

 

 

**Safe stopping point: The purified cDNA can now be stored safely at -20˚C

 

 

Part2:

 

2^nd strand synthesis w/ NEB Next Ultra Directional RNA library kit

 

Make 1^st strand reaction buffer + random primer mix

 

4µl 1^st Strand Buffer

1µl Random primer

5µl H2O

10µl

 

2^nd Strand synthesis

 

10µl cDNA purified

10µl 2X 1^st strand buffer + random primer

20µl

 

95˚C 5min, SNAP cool on ice

 

46µl H2O

8µl 2^nd Strand Syn Buffer 10X

4µl 2^nd Strand enzyme mix

2µl dNTP (10mM)

80 total

 

16˚C 1hr

 

Purify with 1.8X beads, elute with 10µl H2O, and proceed to RNA synthesis

 

 

 

T7 RNA synthesis:

 

10µl purified 2^nd strand cDNA

2µl 10X reaction buffer

2µl ATP

2µl GTP

2µl UTP

2µl CTP

2µl T7 polymerase

22µl

 

37˚C overnight, run 2µl on gel to confirm RNA

 

 

PartIII:

 

DNaseI Digestion:

 

– Add 1µl DNase I to each RNA syn. Reaction. Incubate 37˚C 15min.

 

 

100µl Reaction + H2O

150µl Kapa Beads

 

– purify with 1.5X Kapa Beads. Elute with 20µl H2O

 

RNA Fragmentation

 

Half of our synthetic RNAs will be fragmentized before processed further. The other half will stay unfragmentized.

 

9µl RNA purified

2µl RNA Fragmentation Buffer

9µl H2O

20µl

 

94˚C 2min, SNAP cool on ice

 

add 2µl RNA stop solution

 

Purify with Ampure beads with Isopropanol

 

22µl Fragmentized RNA

28µl H2O

150µl Ampure beads

50µl Isopropanol

 

elute with 10µl H2O , proceed to 3’ adaptor ligation

 

 

 

 

3’ adaptor ligation with NEB small RNA kit

 

Now we have two sets of samples: fragmentized (LRNA enriched) and unfragmentized (sRNA enriched)

 

 

The unfragmentized RNA will be enriched in small RNA, whereas the fragmentized RNA will be mostly large RNA

 

 

• First, dilute MARs 3’adaptor adenylated 6 fold 1µl stock:5µl H2O

 

6µl Input RNA

1µl MARs 3’ adaptors adenylated 6 fold dilution

7µl total

 

70˚C 2min, SNAP cool on ice

 

10µl 3’ ligation reaction buffer 2X

3µl 3’ ligation enzyme mix

20µl

 

incubate 25˚C 1hr

 

 

Hybridize RT Primer:

 

To the ligation reaction: Add:

 

4.5µl H2O

1µl 2^nd RT primer (6 fold dilution of 100µm)

75˚C 5min, 37˚C 15min, 25˚C 15min, 4˚C hold

 

 

 

RT:

Add:

4.5µl H2O

8µl 1^st strand syn buffer

1µl RNaseIn

1µl Protoscript II RT

40µl total

Incubate 50˚C 60min

 

PCR

Add:

 

50µl Long Amp Taq 2X MM

2.5µl universal primer

2.5µl Index primer ( use different Index, carefully note the index number )

5µl H2O

100µl

 

Stage1:

 

94˚C 30s

 

Stage2:

94˚C 15s

62˚C 30s

70˚C 30s

cycle Stage2 for 15 cycles

 

Stage3:

70˚C 5min

4˚C hold

 

run 5µl on gel, add more cycles if necessary

 

 

Safe stopping point: the PCR reaction could be stored in -20˚C until it can be processed further

 

 

Size Selection:

 

• The PCR-adaptor dimer is 150bp, so the library should be size selected from 170bp – 600bp by methods of choice, e.g. e-gel, pippin prep, gel cutting. Concentrate the size-selected library in 20µl NF-H20.
• Quantify with Qubit DNA HS.
• Run 1ng/µl dilution on Agilent Bioanalyzer DNA HS for QC, make sure there is no adaptor dimers left.

 

 

 

 

Sample Sequencing on NextSeq500:

• Pool samples together by equal molar mass, make sure all samples have different index numbers.
• Sequence on illumina NextSeq500 with the following parameters:
  • Use NextSeq500 mid output Kit 150 cycles
  • read 1: 101  , read2: 51 , Index: 6
  • 3pM of library input , while keeping 50% of 1.8pM phiX