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Dunnart ChIP-seq experiment

Experimental design

H3K4me3

  • Signature of active promoters.
  • Closely linked with TSSs.
  • Active, and prefers promoters to enhancers

H3K27ac

  • Signature of active and poised enhancers
  • Active, and prefers enhancers to promoters

Pouch young heads were pooled to generate 2 replicates

collection date PY ID weight (g) head_shape maternal paternal litter sex replicate pool
17/09/2018 Fr113.1 0.014 flat head Fr113 My76 1 A
17/09/2018 Fr113.2 0.016 flat head Fr113 My76 1 A
17/09/2018 Fr113.3 0.0145 flat head Fr113 My76 1 A
17/09/2018 Fr113.4 0.0162 flat head Fr113 My76 1 A
14/11/2018 Fb148.1 0.016 round head Fb148 Mg120 2 B
14/11/2018 Fb148.2 0.016 round head Fb148 Mg120 2 B
14/11/2018 Fb148.3 0.016 round head Fb148 Mg120 2 B
14/11/2018 Fb148.4 0.016 round head Fb148 Mg120 2 B
9/01/2019 Fg123.1 0.0155 flat head Fg123 Mr89 3 B
9/01/2019 Fg123.2 0.0155 flat head Fg123 Mr89 3 B
9/01/2019 Fg123.3 0.0155 flat head Fg123 Mr89 3 B
9/01/2019 Fg123.4 0.0155 flat head Fg123 Mr89 3 B
9/01/2019 Fg123.5 0.0155 flat head Fg123 Mr89 3 B
9/01/2019 Fg123.6 0.0155 flat head Fg123 Mr89 3 B
16/01/2019 Fb148.5 0.014 flat head Fb148 Mg120 4 A
16/01/2019 Fb148.3 0.015 flat head Fb148 Mg120 4 A
16/01/2019 Fb148.4 0.015 flat head Fb148 Mg120 4 A
16/01/2019 Fb148.1 0.015 flat head Fb148 Mg120 4 A
16/01/2019 Fb148.7 0.014 flat head Fb148 Mg120 4 A
16/01/2019 Fb148.2 0.014 flat head Fb148 Mg120 4 A
16/01/2019 Fb148.6 0.014 flat head Fb148 Mg120 4 A
24/09/2019 Fw263.1 0.0164 round head Fw263 Mw270 5 A
24/09/2019 Fw263.2 0.015 round head Fw263 Mw270 5 A
24/09/2019 Fw263.3 0.0157 round head Fw263 Mw270 5 A
24/09/2019 Fw263.4 0.0176 round head Fw263 Mw270 5 A
24/09/2019 Fw263.5 0.0176 round head Fw263 Mw270 5 A
24/09/2019 Fw263.6 0.0146 round head Fw263 Mw270 5 A
24/09/2019 Fw263.7 0.0173 round head Fw263 Mw270 5 A
23/10/2019 Fb197.1 0.0137 flat head Fb197 My234 6 M B
23/10/2019 Fb197.2 0.0112 flat head Fb197 My234 6 M B
23/10/2019 Fb197.3 0.0132 flat head Fb197 My234 6 M B
23/10/2019 Fb197.4 0.0114 flat head Fb197 My234 6 M B
23/10/2019 Fb197.5 0.0098 flat head Fb197 My234 6 F B
23/10/2019 Fb197.6 0.0121 flat head Fb197 My234 6 F B
23/10/2019 Fb197.7 0.0118 flat head Fb197 My234 6 F B
23/10/2019 Fb197.8 0.0116 flat head Fb197 My234 6 F B
23/10/2019 Fb197.9 0.0115 flat head Fb197 My234 6 F B
29/10/2019 Fb255.1 0.0186 round head Fb255 My234 7 F B
29/10/2019 Fb255.2 0.0157 round head Fb255 My234 7 F B
29/10/2019 Fb255.3 0.019 round head Fb255 My234 7 M B
29/10/2019 Fb255.4 0.0187 round head Fb255 My234 7 F B
29/10/2019 Fw264.1 0.0182 round head Fw264 My234 8 F A
29/10/2019 Fw264.2 0.0185 round head Fw264 My234 8 F A
29/10/2019 Fw264.3 0.0168 round head Fw264 My234 8 M A
29/10/2019 Fw264.4 0.0187 round head Fw264 My234 8 M A
19/11/2019 Fb148.1 0.017 round head Fb148 My234 9 F B
19/11/2019 Fb148.2 0.018 round head Fb148 My234 9 M B
19/11/2019 Fb148.3 0.019 round head Fb148 My234 9 F B
19/11/2019 Fb148.4 0.018 round head Fb148 My234 9 M B
27/11/2019 Fb198.1 0.013 flat head Fb198 My234 10 F A
27/11/2019 Fb198.2 0.016 flat head Fb198 My234 10 F A
27/11/2019 Fb198.3 0.012 flat head Fb198 My234 10 F A
27/11/2019 Fb198.5 0.015 flat head Fb198 My234 10 M A


Experiment performed according to manufacturers instructions for the MAGnify™ Chromatin Immunoprecipitation System (492024).

H3K4me3 antibody (ab8580) - https://www.abcam.com/histone-h3-tri-methyl-k4-antibody-chip-grade-ab8580.html

H3K27ac antibody (ab4729) - https://www.abcam.com/histone-h3-acetyl-k27-antibody-chip-grade-ab4729.html

DNA fragments isolated after pull down were sequenced by GENEWIZ (China). Paired-end sequencing at a depth of 40 million reads.


sample ID replicate pool pull down concentration (ng/uL) amount (ug)
A1 A input control 0.39 58.5
A2 A H3K4me3 0.258 38.7
A3 A H3K27ac 1.61 241.5
B1 B input control 0.295 44.25
B2 B H3K4me3 0.242 36.3
B3 B H3K27ac 2.33 349.5


Sequencing

Library construction and sequencing performed by GENEWIZ. Raw data (Pass Filter Data) was processed by adapter trimming and low quality read removal using NGS quality control software Cutadapt (v1.9.1) to generate clean data for subsequent analysis. software:Cutadapt(version 1.9.1) The process includes the following steps:/ 1. remove the adapter sequences; 2. remove the 5 ‘or 3’ end bases of quality scores below 20; 3. remove the reads in which ‘N’ is above 10%; 4. remove reads that are less than 75 bp long after trimming.

The statistics of raw data is summarized in the table below.

Table. Raw data statistics

Sample length Reads Bases Q20(%) Q30(%) GC(%) N(ppm)
A-1 150.00 130587134 19588070100 97.95 95.35 36.18 1.79
A-2 150.00 103324742 15498711300 96.83 93.28 51.24 1.65
A-3 150.00 131071676 19660751400 97.04 93.71 50.57 1.67
B-1 150.00 111574640 16736196000 97.27 93.12 36.35 0.94
B-2 150.00 114146802 17122020300 95.59 89.93 52.60 0.90
B-3 150.00 104714846 15707226900 95.89 90.50 50.02 0.88

The statistics of processed data is summarized in the table below.

Table. Filtered data statistics

Sample length Reads Bases Q20(%) Q30(%) GC(%) N(ppm)
A-1 147.44 130494346 19239781217 98.30 95.83 36.06 1.55
A-2 147.09 103188262 15177774139 97.58 94.25 51.19 1.41
A-3 147.00 130913300 19244653341 97.76 94.65 50.54 1.44
B-1 146.92 111529316 16386102716 97.63 93.63 36.16 0.82
B-2 146.50 114021348 16704280185 96.49 91.10 52.51 0.77
B-3 146.92 104607414 15368815013 96.72 91.59 49.94 0.77

Column explain:
(1) Sample: Sample name
(2) length: Average length of the reads
(3) Reads: Read count
(4) Bases: Base count
(5) Q20, Q30: The percentage of bases with quality scores (Qphred) higher than 20 or 30
(6) GC%: The percentage of G+C in the reads
(7) N(ppm): The number of base ‘N’ per million bases.