Antigen

Antigen Class

RNA polymerase

Antigen

RNA polymerase II

Cell type

Cell type Class

Neural

Cell type

Cerebral Cortex

MeSH Description

Type of cerebral cortex which does not pass through a perinatal phase of six-layered structure as in the NEOCORTEX and develops into three or four layers in the mature brain. Allocortex has three subareas: archi- paleo- and periallo-cortex.

Sample

source_name

RNAPol2Ser5_ChIPSeq

strain

C57BL/6

age

2 months

Sex

male

tissue

Cerebral cortex

chip antibody

RNAPol2Ser5 clone4H8

chip antibody vendor

Abcam

molecule subtype

genomic DNA

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Cortices of 3 mice were homogenized in 1 X PBS including 1% formaldehyde, and the homogenates were kept for 10 min at 25°C. Cross-linking reactions were stopped by the addition of glycine (125 mM) for 10 min at 25°C. Fixed chromatin samples were then homogenized in cell lysis solution (PIPES 5 mM (pH 8), KCl 85 mM, NP40 0.5%) and centrifuged for 5 min at 3000 rpm, 4°C. Pellets were resuspended in RIPA-light solution (NaCl 150 mM, SDS 0.3%, Tris-HCl 50 mM (pH 8)) and sonicated using a covaris E220. Sonicated chromatin samples were then centrifuged for 15 min at 14000 rpm, 4°C. Pellets were resuspended in 1 ml of RIPA-light solution. Chromatin samples were pre-cleared with 50 ul of dynabeads protein G (Life Technologies) pre-blocked with BSA and incubated overnight at 4°C with antibodies anti- RNAPolII phosphorylated in serine 5 (clone 4H8) (Abcam, ab5408). Antibodies and chromatin were then mixed with 100 ul of dynabeads protein G for 3 hours at 4°C. The beads were then washed with RIPA-light solution, washing-solution (Tris-HCl 100 mM (pH 8), LiCl 500 mM). Protein-DNA complexes were eluted from the beads, de-cross-linked, treated with proteinase K and purified. The DNA concentration was determined by fluorometry on the Qubit system (Invitrogen) A total of 100 ng RNAPolII-immunoprecipated DNA was used for the preparation of the library. The immunoprecipitated DNA and input DNA were sheared a second time with the Covaris E220 instrument in 53µl reaction volume (duty factor 10%, Pic Incident Power 175, Cycles per burst 200, time 360 sec) to obtain fragments in the size range of 150 bp followed by purification with AMPure XP beads (x1.8v/v) (Beckman Coulter A63881). Purified DNA was resuspended in 45 µl elution buffer. A library of the chromatin immunoprecipitated DNA fragments was prepared using the TruSeq DNA Low Throughput Protocol (Illumina). Bisulfite conversion was performed using the EZ DNA Methylation-Gold Kit (Zymo Research). PCR enrichment of ligation products was performed using the Illumina Primer Cocktail; 15 cycles of PCR were performed for ChIP libraries and 10 cycles for the input. The libraries were purified using AMPure XP beads x1.0 v/v. Quality of libraries was validated by 260 nm absorbance measurement, quality control on HSdna chip (Agilent Bioanalyzer : size of libraries around 275bp bp) and quantification by Q-PCR with Kappa Library Quantification kit for Illumina Sequencing Platforms (KAPPA Biosystems). The DNA concentration of the different sequencing libraries was from 40 to 500 nM. Clusters (13.5 pM) were generated using TruSeq PE Cluster Kit v3, for cBot protocol, which was followed by Pair End Sequencing, 50 cycles, on an Illumina HiSeq 2000, according to the manufacturer recommendations.

Sequencing Platform

instrument_model

Illumina HiSeq 2000

mm10

Number of total reads

44996499

Reads aligned (%)

98.1

Duplicates removed (%)

3.5

Number of peaks

967 (qval < 1E-05)

mm9

Number of total reads

44996499

Reads aligned (%)

98.0

Duplicates removed (%)

3.7

Number of peaks

927 (qval < 1E-05)