Download PCR PCR conditions and primers used in this study were as follows

PCR
PCR conditions and primers used in this study were as follows:
To obtain the full-length mouse D3 cDNA, total RNA extracted from brain of B6 mouse using
the TRIzol reagent (Invitrogen, Carlsbad CA, USA) were reverse-transcribed into cDNA using
SuperScript II Reverse Transcriptase (Invitrogen). The open reading frame of D3 was
synthesized by amplification of the 5′- part (472 bp) and 3′- part (940 bp) separately by PCR
followed by the ligation. Primers for retroviral vector construction and preparation were follows:
5′- part of mouse D3 cDNA
sense
antisense
3′- part of mouse D3 cDNA
sense
antisense
5′-GCATTACCATGGCACCTCTGA-3′
5′-ACACAGCCGTAATCATGAGCG-3′
5′-CCAGTTCACTATCAGCATGGC-3′
5′-TTCTCGAGTCAGCAGGATAGA-3′
Reactions containing 2.5 U of Taq HS polymerase (Takara Bio, Shiga, Japan) were performed for
5 minutes at 94°C followed by 35 cycles for 30 seconds at 94°C, 30 seconds at 57°C, 1 minute at
72°C, and 1 cycle for 7 minutes at 72°C for amplification of the 5′- part, and for 5 minutes at
94°C followed by 35 cycles for 1 minute at 94°C, 1.5 minutes at 55°C, 2 minutes at 72°C, and 1
cycle for 7 minutes at 72°C for amplification of the 3′- part.
Total RNA from B lymphoma and splenic B cells was isolated using TRIzol reagent (Invitrogen)
followed by synthesis of cDNA using SuperScript II. One hundred nanogram of cDNA was used
for RT-PCR using the PCR Master Mix (Promega, Madison, WI, USA) and primer sets designed
following a program in the Primer Bank (http://pga.mgh.harvard.edu/primerbank/). Primers for
RT-PCR analysis were follows:
Mouse D3
sense
antisense
Mouse β-actin
sense
antisense
Mouse Pax5
sense
antisense
Mouse p50
sense
antisense
Mouse p65
sense
antisense
Mouse c-Rel
sense
antisense
Mouse Oct-2
sense
antisense
Mouse CIITA
sense
antisense
Mouse Blimp-1
sense
antisense
Mouse Bcl-xL
sense
5′-CCTCTGAGCCAGATAAGCAGC-3′
5′-AGACCGTTGCCAAAGATGATG-3′
5′-AAGAGCTATGAGCTGCCTGA-3′
5′-ACGGATGTCAACGTCACACT-3′
5′-AAAGGTTGCCACTCCCAAAGT-3′
5′-GCCTGTAGACACTATGCTGTGAC-3′
5′-CAACGCCCTTTTCGACTAC-3′
5′-GATCCCTCACGAGCTGAGC-3′
5′-TTCCTGGCGAGAGAAGCAC-3′
5′-AAGCTATGGATACTGCGGTCT-3′
5′-AGAGGGGAATGCGGTTTAGAT-3′
5′-TTCTGGTCCAAATTCTGCTTCAT-3′
5′-ACACAGGCTATGACTCGCC-3′
5′-GGAGGGGCTTGAGTTTACACA-3′
5′-CTAGCCAAGTCCCTCCTAAGG-3′
5′-ATGTCAGACTGATCCTGGCAT-3′
5′-TTCTCTTGGAAAAACGTGTGGG-3′
5′-GGAGCCGGAGCTAGACTTG-3′
5′-TGGAGGTAAACTGGGGGTCGCATCG-3′
antisense
5′-AGCCACCGTCATGCCCGTCAGG-3′
Reactions were performed for 2 minutes at 50°C, 1 cycle of 10 seconds at 95°C followed by 27
cycles of 15 seconds at 95°C, 30 seconds at 60°C, 30 seconds at 72°C, and 1 cycle of 10 minutes
at 72°C. The amplification program for Bcl-xL consisted of 1 cycle of 5 minutes at 94°C, 30
cycles of 1 minute at 94°C, 1 minute at 64°C, 1 minute at 72°C, and 1 cycle of 7 minutes at
72°C.
Primer sets for quantitative RT-PCR were follows:
Mouse D3
sense 5′-GCATTACCATGGCACCTCTGA-3′
anisense 5′-TCCTCGAGTCAGCAGGATAGA-3′
Mouse β-actin
sense 5′-ACACTGTGCCCATCTACGAGG-3′
antisense
5′-ACGCTCGGTCAGGATCTTCAT-3′
Quantitative RT-PCR was performed according to the manufacturer’s recommended PCR
condition using ABI PRISM 7900HT (Applied Biosystems).
Primers for clonality analysis were follows:
sense 5′-ACAAGCTTCAAAGCACAATGCCTGGCT-3′
D-JH4
antisense5′-CTCTCAGCCGGCTCCCTCAGGG-3′
7183-JH4
sense 5′-GCAGCTGGTGGAGTCTGG-3′
antisense
5′-CTCTCAGCCGGCTCCCTCAGGG-3′
sense 5′-TCCAGACTGAGCATCAGCAA-3′
Q52-JＨ4
antisense
5′-CTCTCAGCCGGCTCCCTCAGGG-3′
J558-JＨ4
sense 5′-CAGGTCCAACTGCAGCAG-3′
antisense
5′-CTCTCAGCCGGCTCCCTCAGGG-3′
Reactions containing PCR Master Mix (Promega) were incubated for 1 minute at 94°C followed
by 30 cycles of 1 minute at 95°C, 30 seconds at 63°C, 1.5 minutes at 72°C, and 1 cycle of 7
minutes at 72°C.
Primers for linker amplification-mediated PCR (LAM-PCR) analysis were follows:
Biotinylated primer
5′-AGCTGTTCCATCTGTTCTTGGCCCT-3′
Linker primer
5′-GACCCGGGAGATCTGAATTC-3′
Vector-specific primer
5′-GACCTTGATCTGAACTTCTC-3′
Inner primer pair
sense
5′-TCCATGCCTTGCAAAATGGC-3′
antisense
5′-GATCTGAATTCAGTGGCACAG-3′
Juxtaposed sequences of provirus integration were firstly amplified by primer extension using
genomic DNA from transgenic mice, the biotinylated primer, Taq HS (Takara Bio). Biotinylated
PCR products were purified using Dynabeads M-280 streptavidin (Invitrogen). Klenow
polymerase, random primer, dNTP mix (Takara Bio) were added followed by TasI (TOYOBO,
Osaka, Japan) digestion. The samples was ligated with a linker primer, then amplified by the
nested PCR using a vector-specific primer, the linker primer, and an inner primer pair. The final
products were separately isolated by agarose electrophoresis and cloned into the pCR2.1 vector
(Invitrogen).
Microarray-based gene expression profiling
Total RNA was extracted from two cell lines that were independently established from mice
developing the lymphoma (L1 and L2) and splenocytes from two B6 mice as controls (S1 and
S2) using the AllPrep Mini kit (Qiagen, Hilden, Germany). Labeled complementary RNA was
prepared from 0.2 mg of the total RNA using the Agilent one color labeling protocol (Agilent
Technologies, San Clara, CA). Subsequently, 1.65 µg of complementary RNA was fragmented
and hybridized to Agilent whole mouse genome oligo microarray kit G4122F using
manufacturer’s instructions. Signals were scanned by the Agilent G2505B Microarray Scanner,
and were processed by the Agilent Feature Extraction software version 9.5. The output data were
normalized to the 75th percentile prior to data comparisons.
The genes/transcripts that satisfy the following criteria were considered to be up-regulated in the
lymphoma cell lines compared to the control splenocyte samples: (i) the normalize signal values
are over 100 in both of L1 and L2, and (ii) the fold change values are consistently higher than 2.0
in all of the four comparisons (L1/S1, L1/S2, L2/S1, and L2/S1). Similarly, the genes/transcripts
down-regulated were selected using the following criteria: (i) the normalize signal values are
over 100 in both of S1 and S2, and (ii) the fold change values are consistently lower than 0.5 in
all of the four comparisons. The genes that satisfied the above criteria were sorted in the
numerical order of the log2 ratios calculated as log2 (average of L1 and L2 / average of S1 and
S2). Gene Ontology analysis for the differentially expressed genes was conducted using the
DAVID website.
Table S1.
Table S2.