Antigen

Antigen Class

Unclassified

Antigen

Unclassified

Cell type

Cell type Class

Neural

Cell type

Hippocampus

MeSH Description

A curved elevation of GRAY MATTER extending the entire length of the floor of the TEMPORAL HORN of the LATERAL VENTRICLE (see also TEMPORAL LOBE). The hippocampus proper, subiculum, and DENTATE GYRUS constitute the hippocampal formation. Sometimes authors include the ENTORHINAL CORTEX in the hippocampal formation.

Sample

source_name

Hippocampus

strain

BALB/c

gender

male

tissue

Hippocampus

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Chromatin preparation for ChIP and RNA-IP: Mononucleosomes were prepared from testicular tubules as described (Soboleva et al. 2012). To prepare chromatin from hippocampal tissues, 10 Balb/c male mice (6-10 weeks old) were decapitated and hippocampi were surgically removed into ice cold Hank's Balanced Salt Solution (HBSS, Sigma) buffered with 50mM Hepes pH-7.6 and supplemented with 0.2 mM PMSF and EDTA-free protein inhibitor cocktail (Roche)). Hippocampal slices were homogenised in a Dounce homogeniser with 5-10 strokes using pestle A with 4 ml of ice-cold HBSS. Cells were counted and the subsequent preparation of mononucleosomes was identical to that described above for testicular tubules using 2-5 x 107 cells. To prepare sonicated chromatin fragments (∼ 300 base pairs long) from testis for RNA-IP Seq experiments, purified nuclei were re-suspended in 2ml of sonication buffer (50mM Hepes pH-7.6, 1mMEDTA, 05mM EGTA, 0.1% (w/v) SDS, 0.1% (w/v) sodium deoxycholate, 0.2 mM PMSF, Roche EDTA-free protein inhibitor cocktail) and sonicated for 15 minutes (30 seconds intervals on/off at high settings) at 40C using a Bioraptor Sonicator (Diagenode). After sonication, the sheared chromatin was cleared by centrifugation at 10000 g for 5 minutes, dialysed against 10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.5 mM EGTA, 4% (v/v) glycerol and stored at -80°C. Antibodies used were as follows. Anti-H2A.B.3[1], Anti-H2A.Z[2] , Anti-Smith Antigen [Y12] (abcam (ab) 3138); Anti-H2A (ab18255), anti-H3 (ab1791), anti-RNA PolII (phospho S2) (ab5095), anti-RNA PolII (phospho S5) (ab5131), anti-Rent1 (ab109363), anti-Snrpa1 (ab128937), anti-U1A (ab155054), anti-SPT6 (ab32820), anti-Symplekin (ab80274), anti-SAP18 (ab31748) and anti-Sf3b155 (ab66774), all from Abcam. Anti-DDX39A (PA5-31220, Pierce), anti-sheep-HRP (AP324R; Chemicon), anti-rabbit-HRP (AP322P; Chemicon) [1] Soboleva TA, Nekrasov M, Pahwa A, Williams R, Huttley GA, et al. (2012) A unique H2A histone variant occupies the transcriptional start site of active genes. Nat Struct Mol Biol 19: 25-30. [2] Rangasamy D, Berven L, Ridgway P, Tremethick DJ (2003) Pericentric heterochromatin becomes enriched with H2A.Z during early mammalian development. EMBO J 22: 1599-1607. Chromatin immunoprecipitations were performed with affinity purified H2A.B.3, H2A.Z and H3K36me3. ChIP-seq Libraries were prepared for the first biological replicate using the Sample Prep Kit (Illumina) according to the manufacturer's protocol for single end sequencing of DNA. The second biological replicate used the ChIP-Seq Library Prep (New England Biolabs) with barcodes and paired end adaptors. Quality and concentration of the libraries were assessed on Agilent Technologies 2100 Bioanalyzer and using qPCR with adaptor specific primers (ABI Prism 7900HT) according to Illumina's recommendations. DNA was sequenced on HiSeq 2000 or HiSeq 2500 (rapid run mode) (Illumina) using 100 base pairs single– or paired–end reads. RNA-IP libraries were prepared from these low molecular weight RNA-protein complexes and sequenced using 100 base pair paired–end reads.

Sequencing Platform

instrument_model

Illumina HiSeq 2000

mm10

Number of total reads

215937376

Reads aligned (%)

0.0

Duplicates removed (%)

2.8

Number of peaks

38 (qval < 1E-05)

mm9

Number of total reads

215937376

Reads aligned (%)

0.0

Duplicates removed (%)

2.8

Number of peaks

22 (qval < 1E-05)