CapSeq was performed based on ref. 15 (link) and ref. 66 (link). In brief, 1 µg total RNA isolated from wildtype ovaries was treated with TurboDNase (Thermo Fisher Scientific) and purified using RNA Clean & Concentrator-5 columns (Zymo). 5'-monophosphorylated RNAs were then digested by Terminator Exonuclease enzyme (EpiCentre) and any remaining 5' phosphorylated RNAs were dephosphorylated by treatment with Calf Intestine Alkaline Phosphatase (CIP). Following, 5' caps were removed by treatment with Tobacco Acid Pyrophosphatase (TAP) enzyme (EpiCentre; note: the product has been discontinued, but can be replaced by RNA 5' Pyrophosphohydrolase (RppH) from NEB). 5' linkers were then ligated to the decapped RNA 5' ends and cDNA was generated by reverse transcription using an Illumina-compatible RT primer with eight random 3' nucleotides to allow random priming. The cDNA libraries were amplified by PCR using KAPA HiFi HotStart Realtime Mix (Peqlab) and sequenced on a HiSeq2500 (Illumina).
Protocol detail
CapSeq Protocol for Small RNA Identification
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TurboDNase
RNA Clean & Concentrator-5 columns
Terminator Exonuclease enzyme
Tobacco Acid Pyrophosphatase (TAP) enzyme
RNA 5' Pyrophosphohydrolase (RppH)
KAPA HiFi HotStart Realtime Mix
HiSeq2500
Acid
Alkaline phosphatase
Cdna
Cdna libraries
Enzyme
Exonuclease
Intestine
Nucleotides
Ovaries
Primer
Pyrophosphatase
Reverse transcription
Tobacco
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Variable analysis
independent variables
- Total RNA isolated from wildtype ovaries
- 5'-monophosphorylated RNAs
- 5' caps removed by treatment with Tobacco Acid Pyrophosphatase (TAP) enzyme
- 5' linkers ligated to the decapped RNA 5' ends
- CDNA generated by reverse transcription
- CDNA libraries amplified by PCR
- CDNA libraries sequenced on a HiSeq2500 (Illumina)
- 1 µg total RNA isolated from wildtype ovaries
- RNA treated with TurboDNase (Thermo Fisher Scientific)
- RNA purified using RNA Clean & Concentrator-5 columns (Zymo)
- 5'-monophosphorylated RNAs digested by Terminator Exonuclease enzyme (EpiCentre)
- Remaining 5' phosphorylated RNAs dephosphorylated by treatment with Calf Intestine Alkaline Phosphatase (CIP)
- Positive control: Not specified
- Negative control: Not specified
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