Testing NEBNext Low Input RNA Library Prep

Product: Zymo Pico Methyl Seq Library Prep

I am basing my test off of Jill’s protocol, and you can also see Zymo’s protocol here and the general Putnam Lab protocol here.

Here’s the Pico Methyl-Seq library prep workflow:

Materials

• Kit contents
• PCR tubes
• Thermocycler
• Heating block
• Mini centrifuge
• Vortex
• Aluminum beads (to keep things on ice)
• Magnetic stand for PCR tubes
• 1.5 mL tubes
• Shaker

Kit contents

Pico Methyl-Seq™ Library Prep Kit	D5456 (25 preps)	Storage Temperature
Lightning Conversion Reagent	3 tubes	Room Temp.
M-Binding Buffer	20 ml	Room Temp.
M-Wash Buffer*	6 ml (conc.)	Room Temp.
L-Desulphonation Buffer	10 ml	Room Temp.
DNA Elution Buffer	2 ml	Room Temp.
PrepAmp Polymerase (13 U/μL)	15 ul	-20 °C
PrepAmp Buffer (5X)	75 µl	-20 °C
PrepAmp Primer (40 μM)	30 µl	-20 °C
PrepAmp Pre-Mix	120 µl	-20 °C
DNase/RNase-Free Water	1 ml	Room Temp.
Zymo-Spin™ IC Columns	100	Room Temp.
Collection Tubes	100	Room Temp.
DNA Binding Buffer	25 ml	Room Temp.
DNA Wash Buffer	6 ml (conc.)	Room Temp.
LibraryAmp Master Mix (2X)	625 µl	-20 °C
LibraryAmp Primers (10 μM)	30 µl	-20 °C
Index Primer Sets - 6 Sets(10 μM)	30 µl	-20 °C

Protocol

See Jill: https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md

Samples: Starting library prep 8/28/24

Just going to start with two, and then will try again and see if I need to pool RNA or anything.

• POR:
  • #14, 7/24 Charm Extraction
  • Concentration: 1.68 ng/uL
  • 
• POC:
  • #22, 7/30 Zymo Extraction
  • Concentration: 2.55 ng/uL
  • 

https://docs.google.com/spreadsheets/d/1b1TPzleqo81ZLjrh_UIpXEftsjxhxqaMHd7ldFU9oLU/edit?usp=sharing

Protocol notes

Samples were diluted to 1 ng or 10 ng in 20 uL of DNA elution buffer to test ideal input amonts. In section 4, I used 9 cycles for 10 ng input and 12 cycles for 1 ng input samples.

FINISHED SUCCESSFUL LIBRARY PREP

Full results can be found here

sample_id	tapestation concentration	ng Library (conc * 14 uL)
POR_14, 1ng	5.74	80.36
POR_14, 10 ng	10.8	151.2
POC_22, 1 ng	6.36	89.04
POC_22, 10ng	4.62	64.68