darjus

RNA-Seq analysis for Darjus

smRNA seq

1) FASTQC is called to do a quality analysis Find the adapter sequence from the over enriched sequences. Filter reads for quality fastq_quality_filter -q 20 -p 80 -Q 33 -i 101_R1.fastq -o 101 _trim.fastq 2) Trim the reads via cutadapt to remove adapter. cutadapt -a TGGAATTCTCGGGTGCCAAGG -m 22 101_trim.fastq > 101_cut.fastq cutadapt version 1.2.1

Command line parameters: -a TGGAATTCTCGGGTGCCAAGG -m 22 101_trim.fastq

Maximum error rate: 10.00%

No. of adapters: 1

Processed reads: 9112228

Processed bases: 455611400 bp (455.6 Mbp)

Trimmed reads: 9043406 (99.2%)

Trimmed bases: 253078980 bp (253.1 Mbp) (55.55% of total)

Too short reads: 3461749 (38.0% of processed reads)

Too long reads: 0 (0.0% of processed reads)

Total time: 269.30 s

Time per read: 0.03 ms

Adapter 1
Adapter 'TGGAATTCTCGGGTGCCAAGG', length 21, was trimmed 9043406 times. No. of allowed errors:

0-9 bp: 0; 10-19 bp: 1; 20-21 bp: 2 Lengths of removed sequences

length count expected max. errors

3 1699 142378.6 0

4 1538 35594.6 0

5 2328 8898.7 0

6 1364 2224.7 0

7 4477 556.2 0

8 9870 139.0 0

9 12061 34.8 0

10 11305 8.7 1

11 8247 2.2 1

12 10624 0.5 1

13 11304 0.1 1

14 23580 0.0 1

15 27734 0.0 1

16 42759 0.0 1

17 52727 0.0 1

18 56827 0.0 1

19 159030 0.0 1

20 74683 0.0 2

21 40187 0.0 2

22 67283 0.0 2

23 59763 0.0 2

24 66506 0.0 2

25 117030 0.0 2

26 696878 0.0 2

27 996519 0.0 2

28 3025334 0.0 2

29 1714861 0.0 2

30 678956 0.0 2

31 172311 0.0 2

32 100394 0.0 2

33 156367 0.0 2

34 201311 0.0 2

35 218498 0.0 2

36 102742 0.0 2

37 48999 0.0 2

38 22369 0.0 2

39 17709 0.0 2

40 7530 0.0 2

41 4258 0.0 2

42 3497 0.0 2

43 3497 0.0 2

44 2413 0.0 2

45 835 0.0 2

46 508 0.0 2

47 155 0.0 2

48 651 0.0 2

49 118 0.0 2

50 3770 0.0 2

Comparison: Analysis 1 : YAP: Transcriptional co-activator (Yap is low exressed in 3T3 in this cell line) 3T3 -- YAP 3T3 -- Empty Over expression of Yap (retro virus cdna inside and replicates) miRNAs now, earlier we found the genes

Analysis 2: Hepa16- Cell line (Yap is highly expressed in this cell line) Here the Yap is knocked down

Yap Hairpins: 891, 2464, Control : Reni

(So we should try to get overlap between the up regulated in analysis 1 and down regulated in analysis 2)

Analysis3: YAP is Over expressed in Transgenic animals (Invivo validation of the Yap) Livers: 4 numbered samples (Transgenic) (high numbers 4 numbered samples (Controls with lower numbers)

Overlap between the two analyses: Analysis 2 and Analysis 3 (In the opposite direction though, so between up in 2 vs down in 3, we will find overlap since both are liver cell and samples) Small RNA-Seq

1) Livers: with each other (4 replicates vs Controls) Control vs transgenic

2) YAP: Compare 3t3 vs empty

3) 891 vs renilla and 2464 vs renilla

FINALLY:

Take the targets for microRNA's that are Up regulated possible in Livers and find their targets in longRNA's that we did for Yap hairpin samples