Usage Guide

Core facility

◆Usage Guide for shared research equipment

① iSAL equipment (open to both inside and outside KU): iSAL/KUMaCo

10x Genomics Chromium Controller
10x Genomics Chromium X
Bio-Rad QX200 Droplet Digital PCR system
Takara Bio USA SMARTer ICELL8 cx Single-Cell System
Nanopore PromethION24

Bio-Rad C1000 Touch Thermal Cycler (No charge)

Please book the equipment at KUMaCo
※To access to the booking system, you first need to register at KUMaCo.
(KUMaCo registration must be renewed each year)

②ASHBi only equipment: ASHBi Share-e

To apply for an account, please send an email to the address below with your group’s PI in the CC.
(To: ashbi-signac-office【@】mail2.adm.kyoto-u.ac.jp)
※Please replace “【@】” with “@”.
When using the equipment for the first time, we will provide necessary training.

Price List

Click here for a list of usage fees

SignAC open hours: Weekdays 9:00-17:00

◆Usage Guide for analysis services

ASHBi SignAC provides the following analysis services, which are available to researchers both inside and outside KU.

How to request

1. If you request an analysis service for the first time, please contact SignAC by e-mail in advance.
Please specify whether your request is for RNA-Seq, Illumina NGS analysis, or other consultation.
(To: ashbi-signac-office【@】mail2.adm.kyoto-u.ac.jp)
※Please replace “【@】” with “@”.

2. If we accept your request, we will provide you with an account for our service management system (SignAC Redmine).
Please describe details about your samples and requests.

3. At the same time, please make your request through iSAL/KUMaCo of Kyoto University Medical Research Support Center.

  • KUMaCo (You first need to register at KUMaCo before using our services. Registration is required every year.)
※If you are already using SignAC Redmine, please proceed with the above requests through Redmine and KUMaCo at the same time.

4. After your request is confirmed by SignAC, please print out the request form created at KUMaCo and bring/send it to us.
You may submit your samples together.

Where to submit your forms and samples: Room101, 1st floor, Faculty of Medicine Bldg. B, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501
Open hours: Weekdays 9:00-15:30

・Illumina NGS Analysis

We have three Illumina NGS systems, NovaSeq6000, NextSeq550, and iSeq100.
After receiving your library, we will perform the NGS run and give you the sequence results.
Please specify the loading concentration to sequence your library.

・The following table is copied from llumina NovaSeq6000 Denature and Dilute Libraries Guide (1000000106351 v03).
Please refer to the table below for the sample concentration of NovaSeq6000.
・The loading concentration for the NextSeq550 is often 1.5-1.8 pM.
・Please refer to the Illumina website for iSeq.

NovaSeq6000 Reference Installation Concentrations
NovaSeq6000 NGS Sample Concentrations for standard workflows

Library type Final Loading Concentration (pM) Library Concentration at Submission (nM)
PhiX 250 1.25
Illumina DNA PCR-free library pool 400-600 2-3
TruSeq DNA PCR-free library pool 175-350 0.875-1.75
DNA PCR-amplified library pool 300-600 1.5-3.0
Single Cell 250-500 1.25-2.5

Nova Seq6000 NGS Sample Concentrations for XP workflows(only for S4 lane sequences)

Library type Final Loading Concentration (pM) Library Concentration at Submission (nM)
DNA PCRfree library pool 115-235 0.575-1.175
DNA PCRamplified library pool 200-400 1.0-2.0
Single Cell 175-275 0.875-1.375

Required sample volume
NovaSeq6000
Pool all samples and adjust the concentration to 5 times the installed concentration.
Please submit the amounts shown below.
SP/S1: 250 μL or more
S2: 450 μL or more
S4: 700 μL or more
S4 lane sequence: 70 μL or more

NextSeq550
Pool all samples and adjust the concentration to either 4 nM, 2 nM, 1 nM, or 0.5 nM.
Please submit the amounts shown below.
4 nM: 15 μL or more
2 nM: 30 μL or more
1 nM: 60 μL or more
0.5 nM: 120 μL or more

iSeq100
Pool all samples and adjust the concentration to 1 nM.
Please submit 15 μL or more.
1 nM: 15 μL or more.

Data transfer
For researchers outside of ASHBi, data (usually fastq files, but raw data is also an option) will be copied to HDD or flash memory.
If you prefer receiving data as fastq files, please submit a sample sheet with the index information.

Please refer to the Illumina website for how to create a Sample Sheet.
●Illumina Experiment Manager
>Download link

>User Guide Link

●Please feel free to download the sample sheet (sample) from the link below.
>Download Sample Sheet (Sample)

>Sample Sheet (Example)

Please make sure that the direction of index 2 is correctly specified.
Reference →>Link

RNA-seq library preparation and NGS analysis

  • We will prepare libraries from 100 ng of total RNA using the following kits.
    NEBNext Poly(A) mRNA Magnetic Isolation Module[E7490]
    NEBNext Ultra II Directional RNA Library Prep Kit [E7760]
    NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) [E6440]
  • In general, we will perform sequencing with the paired-end mode (more than 50 bp at both ends) to have at least 15 million (usually more than 20 million) clusters per sample.
  • The fastq files will be delivered. We don’t perform adapter trimming.

Requirements for RNA to be brought in

Volume: minimum 8 μL
Total RNA amount: minimum 300 ng
Concentration: 10-150 ng/μL, recommended, 50-100 ng/μL

  • We recommend (but not require) DNase treatment.
  • Please measure the concentration of RNA yourself (Qubit, NanoDrop, etc.).
  • SignAC does not measure RNA integrity (RIN) prior to the library preparation. The library preparation kits we use recommend that the total RNA samples have an RIN of 7 or higher. The RIN can be measured with an Agilent Bioanalyzer, etc. Problems caused by the RNA sample itself are at your responsibility.
  • Please submit an Excel or other file with the sample name and its concentration through the SignAC web system(SignAC Redmine)
  • Please give serial numbers to your sample name and write down the numbers on the tube.
  • We prefer not receiving RNA in 0.2 mL individual tubes.
  • We process your samples based on the concentration indicated by the users.

Regarding medical and health research involving human subjects

  • Please have your human research plan approved by the authorities including a description about how to outsource the analysis to SignAC.
  • Please enter the approval number of your human research plan in SignAC Redmine.
  • Please state what SignAC must do according to your human research plan in SignAC Redmine.
  • Note that SignAC may not be able to accept your analysis request if we decide that we cannot comply with it.

Price List

Click here for a price List (analysis service)

Disclaimer

Please refer to Article 15 of the regulations.

Publication

Please acknowledge SignAC, when you use our facilities and services and publish the research results in papers.
(English)
We thank Single-cell Genome Information Analysis Core (SignAC) at WPI-ASHBi, Kyoto University, for their supports.
(Japanese)
京都大学高等研究院ヒト生物学高等研究拠点 (WPI-ASHBi),単一細胞ゲノム情報解析コア (SignAC) の支援に感謝する。

We would appreciate it if you could provide us with a reprint, a copy, or a PDF of your paper. Thank you for your cooperation.

Publications supported by SignAC

Regulations

Link

Contact

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