rna-seq.md

Defining transcribed regions using RNA-seq

• url: "http://www.nature.com/nprot/journal/v5/n2/full/nprot.2009.229.html"

Yeast culture

1

• process: "Preculture of S. pombe"
  • input: "yeast cells picked from a YES agar plate"
  • input: "YES media"
    • volume: "30 ml"
  • container: "flask"
    • size: "50 ml"
  • temperature: "32 °C"
  • time: "overnight"
  • output: "precultured yeast cells"
    • density: "OD 595 <0.5"

2

• process: "Transfer precultured yeast to fresh YES media"
  • input: "1.1-1"
    • volume: "a sufficient volume"
  • input: "fresh YES media"
    • volume: "100 ml"
  • output: "precultured yeast in fresh YES media"
• process: "Incubation"
  • input: "2.1-1" <!--- 2.1-1 indicates the first output of the first process of the step 2, "precultured yeast in fresh YES media" --->
  • output: "yeast culture"
    • density: "OD 595 of 0.2–0.5"
  • temperature: "32 °C"

3

• process: "Separate the culture"
  • input: "2.2-1"
  • container: "Falcon tube"
    • size: "50 ml"
    • #: 2
  • output: "Separated culture"
    • #: 2
• process: "Centrifuge"
  • input: "3.1-1"
  • output: "Pellet and supernatant"
  • time: "2 min"
  • temperature: "25 °C"
  • gravity: "1,500g"

RNA extraction

4

• process: "Discard the supernatant"
  • input: "3.2-1"
  • output: "yeast cells"
• process: "Resuspend in TES"
  • input: "4.1-1"
  • input: "TES"
    • volume: "750 μl"
  • output: "resuspended cells"
  • instrument: "pipette"

5

• process: "Add acidic phenol–chloroform"
  • input: "4.2-1"
    • is_target: true
  • input: "acidic phenol–chloroform"
    • volume: "750 μl"
  • output: "yeast cells in acidic phenol–chloroform"
• process: "Vortex"
  • input: "5.1-1"
  • time: "15 s"
  • output: "yeast cells in acidic phenol–chloroform"

6

• process: "Preheating heat block"
  • input: "heat block"
  • output: "heat block"
    • temperature: "65 °C"

7

• filter: "Vortex sample every 10 mins in 1 hour incubation"
  • loop: 6
    • process: "Place 10 min"
      • input: "5.2-1"
      • output: "yeast cells in acidic phenol–chloroform"
      • temperature: "65 °C"
      • time: "10 min"
    • process: "Vortex"
      • input: "7.1.1-1"
      • output: "yeast cells in acidic phenol–chloroform"
      • time: "10 s"

8

• process: "Place sample on ice"
  • time: "1min"
  • input: "7.1-1"
  • output: "yeast cells in acidic phenol–chloroform"
• process: "Vortex"
  • time: "20s"
  • input: "8.1-1"
  • output: "yeast cells in acidic phenol–chloroform"
• process: "Centrifuge"
  • time: "15 min"
  • gravity: "14,000g"
  • temperature: "4 °C"
  • input: "8.2-1"
  • output: "centrifuged yeast cells in acidic phenol–chloroform"

9

• process: "Prespin yellow phase-lock tube"
  • time: "10 s"
  • gravity: "14,000g"
  • temperature: "RT"
  • input: "yellow phase-lock tube"
    • size: "2 ml"
  • output: "yellow phase-lock tube"

10

• process: "Add acidic phenol–chloroform to phase-lock tube"
  • input: "9.1-1"
    • is_target: true
  • input: "acidic phenol–chloroform"
    • volume: "700 μl"
  • output: "acidic phenol–chloroform in a yellow phase-lock tube"

11

• process: "Transfer aqueous to the phase-lock tube"
  • input: "8.3-1"
    • volume: "700 μl"
  • input: "10.1-1"
    • is_target: true
  • output: "sample in a acidic phenol-chloroform"

12

• process: "Mixing thoroughly by inverting"
  • input: "11.1-1"
  • output: "sample in a acidic phenol-chloroform"
• process: "Centrifuge"
  • time: "5 min"
  • gravity: "14,000g"
  • temperature: "4 °C"
  • input: "12.1-1"
  • output: "centrifuged sample in a yellow phase-lock tube"

13

• process: "Prespin yellow phase-lock tube"
  • time: "10 s"
  • gravity: "14,000g"
  • temperature: "RT"
  • input: "yellow phase-lock tube"
    • size: "2 ml"
  • output: "yellow phase-lock tube"

14

• process: "Add chloroform:isoamyl alcohol to each phase-lock tube"
  • input: "chloroform:isoamyl alcohol"
    • ratio: "chloroform:isoamyl = 24:1"
    • volume: "700 μl"
  • input: "13.1-1"
    • is_target: true
  • output: "chloroform:isoamyl alcohol in a yellow phase-lock tube"

15

• process: "Add aqueous of centrifuged sample to the phase-lock tube"
  • input: "12.2-1"
    • volume: "700 μl"
  • input: "14.1-1"
    • is_target: true
  • output: "aqueous in chloroform:isoamyl alcohol"

16

• process: "Mixing thoroughly by inverting"
  • input: "15.1-1"
  • output: "mixed sample in phase-lock tube"
• process: "Centrifuge"
  • time: "5 min"
  • gravity: "14,000g"
  • temperature: "4 °C"
  • input: "16.1-1"
  • output: "centrifuged sample in phase lock tube"

17

• process: "Prepare EtOH and NaAc"
  • container: "Eppendorf tube"
    • size: "2 ml"
  • input: "EtOH"
    • volume: "1.5 ml"
    • temperature: "ice cold"
    • concentration: "100%"
  • input: "NaAc"
    • volume: "50 μl"
    • concentration: "3 M"
    • ph: "5.2"
  • output: "Eppendorf tube with ice cold 100% EtOH and NaAc"

18

• process: "Transfer aqueous to Eppendorf tube"
  • input: "16.2-1"
    • volume: "500 μl"
  • input: "17.1-1"
    • is_target: true
  • output: "sample in an Eppendorf tube"
• process: "Vortex tube"
  • time: "10 s"
  • input: "18.1-1"
  • output: "sample in an Eppendorf tube"
• filter: "Store or proceed"
  • case: "user decision"
  • when: "store overnight"
    • process: "Precipitate"
      • input: "18.2-1"
      • pause_point: true
      • temperature: "− 20 °C"
      • time: "overnight"
      • output: "sample in an Eppendorf tube"
  • when: "store half an hour"
    • process: "Precipitate"
      • input: "18.2-1"
      • pause_point: true
      • temperature: "− 70 °C"
      • time: "30 min"
      • output: "sample in an Eppendorf tube"
  • else:
    • process: nil

19

• process: "Centrifuge"
  • input: "18.3-1"
  • time: "10 min"
  • gravity: "14,000g"
  • temperature: "RT"
  • output: "sample in an Eppendorf tube"
• process: "Discard the supernatant"
  • input: "19.1-1"
  • output: "pellet in an Eppendorf tube"
• process: "Add EtOH"
  • input: "19.2-1"
    • is_target: true
  • input: "EtOH"
    • volume: "500 μl"
    • temperature: "4 °C"
    • ratio: "EtOH:DEPC water = 70:30"
      • type: "vol/vol"
  • output: "pellet with EtOH"

20

• process: "Spin the tube"
  • input: "19.3-1"
  • time: "1 min"
  • gravity: "14,000g"
  • temperature: "RT"
  • output: "pellet and supernatant in an Eppendorf tube"
• process: "Aspirate most of the supernatant"
  • volume: "almost"
  • input: "20.1-1"
  • output: "pellet in an Eppendorf tube"
• process: "Spin the tube"
  • input: "20.2-1"
  • time: "5 s"
  • output: "pellet and supernatant in an Eppendorf tube"
• process: "Remove rest of the liquid"
  • instrument: "pipette"
  • input: "20.3-1"
    • volume: "rest of the liquid"
  • output: "pellet in an Eppendorf tube"

21

• process: "Air dry"
  • input: "20.1-1"
  • output: "dried pellet"

22

• process: "Add DEPC water"
  • input: "21.1-1"
    • is_target: true
  • input: "DEPC water"
    • volume: "100 μl"
  • output: "pellet with DEPC water"
• filter: "Incubation time"
  • case: "user decision"
  • when: "Incubate 1 min"
    • process: "Incubate 1 min"
      • input: "22.1-1"
      • temperature: "65 °C"
      • time: "1 min"
      • output: "sample in an Eppendorf tube"
  • when: "Incubate 10 min"
    • process: "Incubate 10 min"
      • input: "22.1-1"
      • temperature: "RT"
      • time: "10 min"
      • output: "sample in an Eppendorf tube"
• process: "Dissolve pellet by pipetting up and down"
  • input: "22.2-1"
  • instrument: "pipette"
  • while: "until no particles are left"
  • output: "sample RNA"
• process: "Vortex gently"
  • input: "22.3-1"
  • time: "10 s"
  • output: "sample RNA"

23

• filter: "Instrument for measurement"
  • case: "user decision"
  • when: "Nanodrop"
    • process: "Measure the RNA concentration with Nanodrop"
      • input: "22.4-1"
      • output: "sample RNA"
      • output: "concentration data"
        • unit: "mg"
        • expect: "1.6–4 mg"
  • when: "UV spectrophotometer"
    • process: "Measure the RNA concentration with UV spectrophotometer"
      • input: "22.4-1"
      • input: "DEPC water"
        • is_reference: true
      • output: "sample RNA"
      • output: "concentration data"
        • unit: "OD"
        • expect: "OD 0.2–0.5"
• process: "Store"
  • input: "23.1-1"
  • temperature: "− 70 °C"
  • expiration_time: "indefinitely"
  • output: "Stored sample RNA"
  • skippable: true

RNA purification

24

• filter: "Low purity of RNA"
  • case: "260:230 ratio"
  • when: "low"
    • process: "Purify RNA by RNeasy"
      • input: "23.2-1"
        • volume: "100 μg"
      • kit: "RNeasy spin column"
      • output: "purified sample RNA"
  • else:
    • process: nil
      • input: "23.2-1"
      • output: "sample RNA"

25

• process: "Incubate RNA with RNase-free DNase"
  • kit: "RNase-free DNase"
  • input: "24.1-1"
  • time: "30 min"
  • temperature: "37 °C"
  • output: "sample RNA"

26

• process: "Purify samples using Qiagen RNAeasy columns"
  • input: "25.1-1"
    • volume: "100 μg"
  • kit: "RNeasy spin column"
  • output: "purified sample RNA"
• process: "Store RNA"
  • input: "26.1-1"
  • temperature: "− 70 °C"
  • expiration_time: "indefinitely"
  • output: "stored sample RNA"
  • skippable: true

27

• process: "Assess the quality of the purified RNA"
  • input: "26.2-1"
  • kit: "RNA Nano chip"
  • instrument: "Agilent Bioanalyzer"
  • output: "sample RNA"
  • output: "RNA quality profile"
  • output: "electrophoretogram"
  • output: "RNA integrity number"
    • expect: ">9"

Poly(A) enrichment of RNA

28

• process: "Enrich the RNA sample for mRNA"
  • input: "26.2-1"
  • kit: "poly(A)+ RNA selection kit"
    • example: "Oligotex mRNA purification kit from Qiagen"
  • output: "poly(A)+ mRNA"
• process: "Measure RNA quantity by Nanodrop"
  • input: "28.1-1"
  • output: "poly(A)+ mRNA"
  • output: "RNA quantity"
    • expect: "~25% of input"

cDNA preparation

29

• process: "Reverse transcription"
  • input: "28.2-1"
  • input: "oligo-dT 20 primer"
  • kit: "SuperScript double-stranded cDNA synthesis kit"
  • output: "sample cDNA"
• process: "Purify the cDNAs"
  • input: "29.1-1"
  • kit: "QIAquick PCR purification kit"
  • output: "sample cDNA"
• process: "Store cDNA"
  • input: "29.2-1"
  • temperature: "− 20 °C"
  • expiration_time: "indefinitely"
  • output: "Stored cDNA"
  • skippable: true

30

• process: "Assess the quality of the prepared cDNA"
  • input: "29.3-1"
  • kit: "DNA 1000 kit"
  • instrument: "Agilent Bioanalyzer"
  • output: "sample cDNA"
  • output: "quality of cDNA"

Sample preparation for Illumina 1G machine

31

• process: "Dilute the pooled cDNA sample"
  • input: "30.1-1"
    • is_target: true
  • input: "TE"
    • volume: "50 μl"
  • ipnut: "nebulization buffer"
    • brand: "Illumina"
    • volume: "700 μl"
  • output: "deluted cDNA"

32

• process: "Fragment sample"
  • input: "31.1-1"
  • instrument: "nebulizer"
    • configuration: "size: 32–35 psi"
  • time: "6 min"
  • condition: "on ice"
  • output: "cDNA fragment"

33

• process: "Purify the fragmentation products"
  • input: "32.1-1"
  • input: "water"
    • volume: "50 μl"
  • kit: "QIAquick kit"
  • output: "purified cDNA fragments"

34

• process: "Incubate fragments"
  • input: "33.1-1"
  • input: "Klenow DNA polymerase"
    • volume: "50 U"
  • input: "T4 DNA polymerase"
    • volume: "30 units"
  • input: "10 mM dNTPs"
    • volume: "20 μl"
  • time: "30 min"
  • temperature: "16 °C"
  • output: "cDNA fragments"

35

• process: "Purify fragments"
  • input: "34.1-1"
  • input: "Qiagen elution buffer"
    • volume: "32 μl"
  • kit: "QIAquick PCR purification column"
  • output: "purified cDNA fragments"

36

• process: "Add exo-Klenow"
  • input: "35.1-1"
    • is_target: true
  • input: "exo-Klenow"
    • volume: "15 units"
  • input: "dATP"
    • volume: "10 μl"
    • concentration: "1 mM"
  • input: "Klenow buffer"
    • volume: "5 μl"
    • concentration: "10×"
  • output: "cDNA fragment mix"
• process: "Incubation to add a 3′ adenine overhang to the blunt-ended double-stranded cDNA fragments"
  • input: "36.1-1"
  • temperature: "37 °C"
  • time: "30 min"
  • output: "cDNA fragment with 3' adenine overhang"

37

• process: "Ligate the tailed cDNA fragments to the two proprietary oligonucleotide adaptors"
  • input: "36.2-1"
  • input: "T4 DNA ligase"
    • brand: "NEB, Ipswitch, Massachusetts, USA"
  • kit: "Illumina kit"
  • output: "cDNA fragment with adaptors"
• process: "incubation"
  • input: "37.1-1"
  • temperature: "RT"
  • time: "15 min"
  • output: "cDNA fragment with adaptors"

38

• process: "Separate ligation products on a gel"
  • input: "37.2-1"
  • input: "Tris-acetate-EDTA (TAE)-agarose gel"
    • ratio: "Tris-acetate-EDTA (TAE):agarose = 2:98"
      • type: "wt/vol"
  • instrument: "razor blade"
  • output: "separated gel"
    • size: "120–170 bp"

39

• process: "extract ligation products"
  • input: "38.1-1"
  • kit: "Qiagen Gel purification kit"
  • input: "water"
    • volume: "30 μl"
  • output: "cDNA fragments"

40

• process: "Set up PCR reactions"
  • ipnut: "39.1-1"
    • volume: "1 μl"
  • input: "water"
    • volume: "22 μl"
  • input: "Phusion Taq master mix"
    • concentration: "2X"
    • volume: "25 μl"
  • input: "1.1 primer"
    • volume: "1 μl"
  • input: "2.1 primer"
    • volume: "1 μl"
  • output: "PCR mix"
• process: "PCR"
  • process: "heating"
    • temperature: "98 °C"
    • time: "30 s"
    • input: "40.1-1"
    • output: "heated PCR mix"
  • filter: "18 cycles"
    • loop: 18
      • process: "denaturation"
        • temperature: "98 °C"
        • time: "10 s"
        • input: "40.2.1-1"
        • output: "PCR mix"
      • process: "annealing"
        • temperature: "65 °C"
        • time: "30 s"
        • input: "40.2.2.1-1"
        • output: "PCR mix"
      • process: "extension"
        • temperature: "72 °C"
        • time: "30 s"
        • input: "40.2.2.2-1"
        • output: "PCR mix"
  • process: "cooling"
    • temperature: "72 °C"
    • time: "300 s"
    • input: "40.2.2-1"
    • output: "PCR mix"
  • process: "keep"
    • temperature: "4 °C"
    • expiration_time: "Infinite"
    • input: "40.2.3-1"
    • output: "amplified cDNA fragment"
• process: "Store"
  • temperature: "4 °C"
  • time: "overnight"
  • input: "40.2-1"
  • output: "amplified cDNA fragment"
  • skippable: true

41

• process: "purify the PCR products"
  • input: "40.3-1"
  • output: "purified PCR products"
  • kit: "QIAquick PCR kit"
• process: "measure the concentration of PCR products"
  • input: "41.1-1"
  • output: "purified PCR products"
  • output: "concentration data"
  • instrument: "Nanodrop"

42

• process: "Dilute the cDNA in TE"
  • input: "41.2-1"
  • input: "TE"
  • output: "fragment library"
    • concentration: "10 nM"
• process: "Store"
  • temperature: "− 20 °C"
  • expiration_time: "indefinitely"
  • ipnut: "42.1-1"
  • output: "fragment library"
  • skippable: true

Illumina genome analyzer analysis

43

• process: "load cDNA fragments onto an Illumina flowcell"
  • input: "42.2-1"
  • output: "Illumina machine ready to run"
  • instrument: "Illumina Genome Analyzer"
• process: "Run the ‘on board’ Illumina analysis pipeline"
  • input: "43.1-1"
  • output: "fastq file"
  • instrument: "Illumina Genome Analyzer"

Pending points

• Assigning type of the process e.g. type: centrifuge or type: vortex for easier mapping to automated platform
• How to describe "do further processes for each item" like enumerable
• How to describe "Use Illumina kit"
• How to describe "can stop here" or "store"