Antigen

Antigen Class

Input control

Antigen

Input control

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

hHipp_Input

age

63y

tissue

Hippocampus

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

~100-200 mg tissue from mouse hippocampus/ human hippocampus was cross-linked in Cross-linking buffer (10 mM HEPES, pH 7.2, 100 mM NaCl, 1 mM EDTA and 1 mM EGTA) containing 1% formaldehyde (Thermo; methanol free) for 10 minutes at room temperature. Excess formaldehyde was quenched by glycine for 5 minutes at room temperature. Tissue was resuspended in homogenization buffer (250 mM Sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH; pH 7.8, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, and protease inhibitors) after a wash with ice-cold PBS. Tissue was homogenized using Dounce homogenizer with loose (A) and tight (B) pestle (5 strokes each), followed by additional 5 strokes of tight pestle with 0.3% NP-40, and then the homogenate was passed through a 40 um strainer. Nuclei were isolated at 4000g/ 4°C for 5 minutes and resuspended in ~0.5 ml sonication buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 mM EGTA). Fragmented chromatin was prepared from ~10 million nuclei in Covaris S220 (200 cycle per burst, 5% duty cycle at power level 4 for 12 minutes at 4°C) in presence of 0.3% SDS. For ChIP, Protein G Dynabeads (Invitrogen; 30 ul/reaction) were equilibrated in TBSTBp (TBS with 1% BSA, 1% Triton-X-100, and protease inhibitors). 10 ul Protein G beads were used for preclearing the chromatin in ChIP buffer (10 mM Tris-HCl pH 8.0, 1% Triton-X-100, 150 mM NaCl and 1 mM EDTA, and protease inhibitors) for 4 hours at 4°C. 20 ul Protein G beads were used to make the bead-Ab complex with 20 ug anti-REST-C antibodies (Ballas et al., 2005) for 2 hours at room temperature in 0.5 ml TBSTBp. After washing bead-Ab complex three times with TBSTBp, ChIP was carried out overnight at 4°C with precleared chromatin. Beads were washed twice with low salt, high salt and LiCl buffers at 4°C, followed by two washes with TE at room temperature. DNA was eluted and crosslinks were reversed overnight at 65°C. Contaminants were removed from DNA by RNaseA and Proteinase K treatment followed by purification with PCR purification kit (QIAGEN) Ten nanograms of fragmented DNA was used as input for a modified TruSeq Nano DNA library preparation protocol (Illumina). Briefly, input DNA was treated with 3' to 5' exonuclease activity and 5' to 3' polymerase activity to blunt the ends. There was no size selection of the fragments. A single “A” nucleotide was added to the 3' ends to enhance ligation to the adapters. RNA adapters (Illumina) were ligated to the fragmented DNA followed by cleanup using sample purification beads (SPB) provided with the kit. The ligation product was enriched using 14 cycles of polymerase chain reaction. The amplification product was cleaned using SPB. Libraries were profiled using the TapeStation D1000 DNA tape. Library concentrations were determined using the Library Quantification Kit for Illumina sequencing platforms (Kapa Biosystems) on a StepOnePlus Real Time PCR Workstation (ThermoFisher). Libraries were mixed for multiplexing and the concentration of the mix was determined by real time PCR.

Sequencing Platform

instrument_model

Illumina HiSeq 2500

hg38

Number of total reads

64625393

Reads aligned (%)

97.7

Duplicates removed (%)

3.6

Number of peaks

1960 (qval < 1E-05)

hg19

Number of total reads

64625393

Reads aligned (%)

96.6

Duplicates removed (%)

4.9

Number of peaks

1249 (qval < 1E-05)