Antigen

Antigen Class

Histone

Antigen

H3K27ac

Cell type

Cell type Class

Neural

Cell type

Hypothalamus

MeSH Description

Ventral part of the DIENCEPHALON extending from the region of the OPTIC CHIASM to the caudal border of the MAMMILLARY BODIES and forming the inferior and lateral walls of the THIRD VENTRICLE.

Sample

source_name

Mus Musculus hypothalamus

sort

LepRb - negative

tissue

hypothalamus

treatment

Fasted - 2 hours Leptin

chip antibody

K27ac

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Hypothalami were pooled and homogenized by douncing 30 times in 1 mL lysis buffer (15 mM Tris-HCL (pH 7.5), 0.1 mM EGTA, 15 mM NaCl, 60 mM KCl, 5 mM MgCl2, 300 mM sucrose, 1 mM DTT, 0.1 mM PMSF, 0.15 mM spermine, 0.5 mM spermidine, Complete protease inhibitor (Roche) and 20mM Na-butyrate (Sigma), RiboLock RNase inhibitor) using a tissue grinder type B (Kimble Chase). The cell lysate was centrifuged at 500 g for 5 minutes at 4ºC. Pellet was re-suspended in 1 mL lysis buffer, and equal volume of nuclei isolation buffer (lysis buffer supplemented with NP40 at 0.4%) was added. The lysate was incubated on ice for 5 minutes, followed by centrifugation at 7000 g for 5 minutes at 4ºC. Cell nuclei pellet was re-suspended in 250 uL FACS buffer (0.15 mM spermine, 0.5 mM spermidine, Complete protease inhibitor (Roche) and 20mM Na-butyrate (Sigma), RiboLock RNase inhibitor in PBS). FACS was performed with FACS Aria II (BD Biosciences) equipped with 70 um nozzle. RNA-seq: 50,000-70,000 GFP positive and negative nuclei were sorted directly into buffer RLT supplemented with beta-mercaptoethanol. RNA was extracted using RNeasy mini kit (Qiagen) following the manufacture's protocol. ChIP-seq: 300,000-540,000 GFP positive and negative nuclei were sorted into PBS. After FACS, FACS buffer was added for a final volume of 10 mL. 50 uL of 1M CaCl2 and 30 uL of 1M MgAc2 was added and incubated on ice for 5 minutes, followed by centrifugation at 1800 g for 15 minutes at 4ºC. The supernatant was removed and FACS buffer supplemented with 1% formaldehyde was gently added onto the pellet to crosslink chromatin. The crosslinked pellet was then washed with FACS buffer supplemented with 125 mM glycine without disturbing the pellet. The crosslinked chromatin was lysed in 130 uL of Buffer B (LowCell# ChIP kit, diagenode) supplemented with Complete protease inhibitor (Roche) and 20mM Na-butyrate (Sigma). The lysed chromatin was sheared using a Covaris S2 sonicator to obtain on average 250bp size fragments. 870 uL of Buffer A (LowCell# ChIP kit, diagenode) supplemented with complete protease inhibitor (Roche) and 20mM Na-butyrate (Sigma) was added. 20 uL of the chromatin solution was saved as an input control. A mixture of 40 uL of Dynabeads protein A and 40 uL of Dynabeads protein G was washed twice with Buffer A (LowCell# ChIP kit, diagenode) and resuspended in 800 uL of Buffer A. 10 ug of H3K27ac antibody (ab4729, Abcam) was added to the beads, and gently agitated at 4ºC for 2 hours. The beads-antibody complex was precipitated with a magnet, and the supernatant was removed. 800 uL of shared chromatin was added to the beads-antibody complex and rotated at 4ºC overnight. The immobilized chromatin was then washed with Buffer A three times and Buffer C once, and eluted in 100 uL of IPure elution buffer (IPure kit, diagenode). In addition, 80 uL of IPure elution buffer was added to 20 uL input. DNA was purified using the IPure kit according to manufacture's protocol. ATAC-seq: After FACS, 49 uL of sorted nuclei (11,200-18,000 nuclei) were mixed with 50 uL Tagment DNA buffer (Nextera DNA sample preparation kit, Illumina) and 1 uL of Tagment DNA enzyme (Nextera DNA sample preparation kit, Illumina), followed by incubation at 37ºC for 30 minutes. Tagmented DNA was purified with MinElute reaction cleanup kit (Qiagen). RNA-seq: RNA was quantified with Qubit RNA HS assay kit. cDNA was amplified using Ovation V2 kit (NuGEN) and sequencing libraries were generated using NexteraXT kit (Illumina). ChIP-seq: Sequencing libraries were generated using Accel-NGS 2S Plus DNA library kit (Swift Biosciences). DNA was quantified with Qubit DNA HS assay kit and Bioanalyzer (Agilent) using the DNA High Sensitivity kit. ATAC-seq: After FACS, 49 uL of sorted nuclei (11,200-18,000 nuclei) were mixed with 50 uL Tagment DNA buffer (Nextera DNA sample preparation kit, Illumina) and 1 uL of Tagment DNA enzyme (Nextera DNA sample preparation kit, Illumina), followed by incubation at 37ºC for 30 minutes. Tagmented DNA was purified with MinElute reaction cleanup kit (Qiagen). The DNA was size-selected using SPRIselect (Beckman Coulter) according to manufacture's double size selection protocol. DNA:SPRIselect ratio was 5:3 for right side, and 2:3 for left side selection.

Sequencing Platform

instrument_model

Illumina HiSeq 4000

mm10

Number of total reads

65283104

Reads aligned (%)

50.0

Duplicates removed (%)

12.2

Number of peaks

510 (qval < 1E-05)

mm9

Number of total reads

65283104

Reads aligned (%)

49.9

Duplicates removed (%)

12.2

Number of peaks

559 (qval < 1E-05)