strain/background: BALB/c gender: female age: 4 weeks cell type: splenocytes infection: HSV-1 mutant strain M3 time point: day 60
Treatment protocol
The HSV-1 mutant strain M3 or wild type strain Mckrae infected BALB/c mice and uninfected control mice were sacrificed at indicated time points post-infection (day 7/ day 30/ day 60). The splenocyte of the mice were homogenated into single cells.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted and purified using miRNeasy Mini Kit (Cat#217004, QIAGEN, GmBH, Germany) following the manufacturer’s instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, US).
Label
Cy3
Label protocol
Total RNA was amplified and labeled by Low Input Quick Amp Labeling Kit, One-Color (Cat.# 5190-2305, Agilent Technologies, Santa Clara, CA, US), following the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat.# 74106, QIAGEN, GmBH, Germany).
Hybridization protocol
Each slide was hybridized with 600 ng Cy3-labeled cRNA using Gene Expression Hybridization Kit (Cat.# 5188-5242, Agilent Technologies, Santa Clara, CA, US) in Hybridization Oven (Cat.# G2545A, Agilent Technologies, Santa Clara, CA, US), according to the manufacturer’s instructions. After 17 hours hybridization, slides were washed in staining dishes (Cat.# 121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit (Cat.# 5188-5327, Agilent Technologies, Santa Clara, CA, US), followed the manufacturer’s instructions.
Scan protocol
Slides were scanned by Agilent Microarray Scanner (Cat#G2565CA, Agilent Technologies, Santa Clara, CA, US) with default settings, Dye channel: Green, Scan resolution=3μm, PMT 100%, 20bit. Data were extracted with Feature Extraction software 10.7 (Agilent Technologies, Santa Clara, CA, US).
Description
M3-day60-3 Gene expression of M3 infected mice splenocytes at day 60 post-infection.
Data processing
The scanned images were analyzed with Feature Extraction Software 10.7 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Raw data were normalized by Quantile algorithm, limma packages in R.
Submission date
Dec 24, 2017
Last update date
Jan 23, 2018
Contact name
Xiaolong Zhang
Organization name
Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College
