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Getting started

Materials

Reagents
REAGENT SOURCE IDENTIFIER
Dra I enzyme NEB Cat#R0129
Alkaline Phosphatase Calf Intestinal (CIAP) enzyme Promega Cat#M1821
Exonuclease I enzyme NEB Cat#M0293
NaOH solution for molecular biology 10 M in H2O Sigma Cat#72068
UltraPure™1M Tris-HCl pH7.5 Invitrogen™ Cat#15567027
Tissue-Tek OCT Sakura Finetek Cat#4583
Methanol (min. 99.8%) Th. Geyer Cat#1437
2-propanol (min 99.9%) Th. Geyer Cat#1197
Mayer’s Haematoxylin Agilent Dako Cat#S3309
Bluing buffer Agilent Dako Cat#CS702
Eosin Y, aqueous Sigma Cat#HT110216
Pepsin from porcine gastric mucosa Sigma Cat#P7000
20x SSC Sigma Cat#S6639-1L
Hydrochloric Acid (HCl) 10N AppliChem Cat#187051
BSA Molecular Biology Grade (conc. 20 mg/ml) NEB Cat#B9000S
dNTP SET 100mM 4X1mL Life Technologies Cat#R0182
SuperScript IV Reverse Transcriptase Life Technologies Cat#18090010
RiboLock RNase Inhibitor Thermo Scientific Cat#EO0381
Tris-HCl Buffer pH 8.0, 1M Life Technologies Cat#AM9855G
Sodium chloride NaCl (5M), RNase-free Invitrogen Cat#AM9760G
Roti®-Stock 20 % SDS ready-to-use, sterile filtered Roth Cat#1057.1
UltraPure 0.5M EDTA, pH 8.0 Life Technologies Cat#15575020
Proteinase K (800 mU/μL) NEB Cat#P8107S
DNA Polymerase Large Fragment exo- Klenow Fragment (3'-5' exo-) NEB Cat#M0212
Ampure XP beads Beckman coulter Cat#A63881
Kapa HiFi Hotstart Readymix KK2612 Roche Cat#7958960001
1.5% Agarose gel, PippinHT (300-1500 bp) Biozym HTC1510
Qubit dsDNA HS Assay Kit Invitrogen Cat#Q32854
High sensitivity DNA kit Agilent Cat#5067-4626
HS RNA tapestation Agilent 5067-5579/5580/5581
Blue S'Green qPCR mix Biozym Cat#331416
KAPA LQ Primer + Mastermix (Illumina/ LC480) Roche Cat#7960573001
KAPA Library Quantification DNA Standards (Illumina) Roche Cat#7960387001
NovaSeq 6000 S4 reagent kit v1.5 (35 cycles) Illumina Cat#20044417
Oligonucleotides
OLIGO SEQUENCE COMMENT
HDMI32-DraI CAAGCAGAAGACGGCATACGAGATTCTTTCCCTACACGACGCTCTTCCGATCTNNVNBVNNVNNVNNVNNVNNVNNVNNVNNNNNTCTTGTGACTACAGCACCCTCGACTCTCGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAAAGACTTTCACCAGTCCATGATGTGTAGATCTCGGTGGTCGCCGTATCATT
Randomer TCAGACGTGTGCTCTTCCGATCTNNNNNNNNN
Read1-DraI ATCATGGACTGGTGAAAGTCTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCGAGAGTCGAGGGTGCTGTAGTCACAAGA
p5_fwd AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCT∗T∗C *denotes phosphorothioated DNA bases
p7_rev_indexing CAAGCAGAAGACGGCATACGAGAT[8-mer index sequence]GTGACTGGAGTTCAGACGTGTGCTCTTCC∗G∗A *denotes phosphorothioated DNA bases
Equipment
  • Chemical hood (for work with toxic chemicals, such as Trizol or methanol)
  • Cryostat
  • Heating block (Can also use hybridisation oven for pre-warming pepsin.)
  • Hybridization oven
  • Brightfield imaging system add objective requirements, camera
  • Thermocycler
  • qPCR machine
  • Bluepippin or PippinHT (alternatively, use manual agarose gel setup and DNA extraction)
  • Automated gel electrophoresis machine (eg. Tapestation or BioAnalyzer)
  • Qubit fluorometer
  • 3D printer (If you don't have a 3D printer, you can check for 3D printing services near you)