N.         Hybrid Mouse Diversity Panel:  F1 hybrids

1.         Mice (L. Martin)
2.         Clinical phenotypes (L. Martin)
3.         RNA-Seq, liver (L. Martin)
4.         Allele-specific expression, Eskin (L. Martin)
5.         RNA-Seq, adipose (Y. Hasin)

 

 

1.         Mice (L. Martin)

In development.

 


2.         Clinical phenotypes (L. Martin)

In development.

 


3.         RNA-Seq, liver (L. Martin)

In development.

 


4.         Allele-specific expression, Eskin (L. Martin)

In development.

 


5.         RNA-Seq, adipose (Y. Hasin)

C57BL/6, DBA and F1 BxD and DxB, males and females. F1 mice were bred at the lab (from Lisa), C57 and DBA are from Jax. All mice were 16 weeks old on normal chow diet at sac. Gonadal fat was flash-frozen in liquid nitrogen and kept at -80C.

Extraction of total RNA from adipose:

In general we use standard Qiazol protocol in conjunction with the Qiagen RNeasy or miRNeasy columns. There are two important modifications though:

1. For homogenization of adipose we use relatively a lot of Qiazol – each fat pad was divided into 3-4 pieces, and each piece was homogenized in 1.5-2ml Qiazol.

2. According to standard Qiazol protocol - after homogenization  samples are incubated at room temperature for  5min (step 4 in Qiazol protocol), then chloroform is added. For adipose samples we use an additional slow centrifugation step after the incubation and before adding chloroform – 3-5min at ~1000-3000g,  room temperature. The homogenate separates into 4 phases:

1- top- transparent liquid – lipids.
2- between qiazol and lipids typically there is a thin debris layer
3- pink qiazol phase (this is where the RNA is)
4– sometimes there is pellet (it should be small, if its large you didn’t homogenize well)

After this centrifugation I transferred the qiazol  phase into a clean tube. Its important to transfer the phase as clean as possible, even if you leave 100-200µl behind.
Add chloroform - 200µl per 1ml of clean phase.
Continue exactly as in Qiagen protocols.
Typically I elute in 40µl of water and get ~300-350 ng/µl of total RNA in the miRNeasy kit.

Preparing samples for sequencing:
In each group (strain and sex) RNA was equimolar pooled from 3 individual fat pads (except F1 BxD females where I had only 2 mice, Table1).

 

f1hybrids_table

 

For each sample miRNA and poly-1 sequencing libraries were prepared by City of Hope – all miRNA libraries were barcoded and sequenced on 1 lane, all polyA libraries were sequenced with paired end.

Mapping and differential expression analysis:
miRNA - reads were de-multiplexed into original libraries (using in house perl scripts) and aligned against the mouse genome (mm9) with two different aligners (Mr/Mrs.Fast and Novoalign, results were highly concordant - Pearson correlation =0.99). Expression was quantitated by counting the number of reads mapping to each mouse miRNA (using mirBase 17 definition).
Alignment of mRNA libraries was done using Mr/Mrs.Fast against the mouse genome (mm9), allowing for up to 3 mismatches in the 50bp reads. Transcript expression was measured by counting the number of reads mapping to each of the transcripts in mm9 RefSeq table (downloaded from UCSC genome browser).

Differentially expressed miRNAs were identified with DEseq R package.
Differentially expressed genes were identified using DeSEQ package,and using cufflinks (for which reads were aligned again with Tophat/Bowtie).