above conditions holds, or if peaks for all three modifications have
not been identified, then a prediction is not made. The region
surrounding the predicted miRNA TSS is defined as the promoter.
Predicted TSSs are expected to be within 500 bp of the actual TSSs
based on the TSS predictions of 1000 highly expressed genes in
resting T cells (Supplemental Fig. S6). Predicted TSSs were manually
inspected to filter out obvious false-positives by taking into ac-
count the three modifications used for prediction, as well as
H3K36me3, H3K79me2, and the coordinates of known genes.
Total RNA from resting and activated CD4+ T cell was prepared
using RNeasy kit (Qiagen). Human placenta RNA was obtained
from Clontech. Reverse transcription was performed using 1 mgof
RNA and a mixture of RT primers (Supplemental Fig. S7A; Sup-
plemental Table S6) using a Smart RACE cDNA amplification kit
(Clontech). PCR was performed using Universal primer (anneals to
Clontech Smart Oligo) and individual RT primer. If no product was
obtained, nested primer was used (Supplemental Table S6).
Promoter reporter assay
Approximately 650 bp fragments surrounding putative promoters
were amplified by PCR using high fidelity Phusion DNA poly-
merase (NEB) and cloned into KpnI/ BglII sites of pGL3 enhancer
vector (Promega). Primers are listed in Supplemental Table S7.
Jurkat cells were plated at 6 3 10
per well of six-well plate and
transfected with 500 ng of reporter construct, 50 ng of pRLTK
Renilla luciferase control plasmid, and 1 mg of carrier DNA (pBSK)
using Superfect reagent (Qiagen). Dual luciferase assay was per-
formed 2 d later, and firefly luciferase signal was normalized by
that of Renilla luciferase.
This work was supported by the Intramural Research Program of
the National Heart, Lung and Blood Institute (NHLBI), National
Institutes of Health. The gene expression analysis using the Affy-
metrix microarrays was performed by the Genomics Core Facility
Author contributions: A.B., R.J., S.C., and K.Z. designed the
study and wrote the paper; A.B., S.C., K.C. and T-Y.R. performed the
experiments; R.J. analyzed the data with contributions from A.B.
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