Random p dn 6 primers

Total RNA from the hypothalamus was extracted with TRIzol reagent (Invitrogen). Assessment of RNA quantity and quality was performed with an Epoch Microplate Spectrophotometer (Biotek). Total RNA was incubated with DNase I RNase-free (Roche Applied Science). Reverse transcription was performed with 2 µg of total RNA with SuperScript II Reverse Transcriptase (Invitrogen) and random primers p(dN)6 (Roche Applied Science). Real-time polymerase chain reaction was performed using the 7500TM Real-Time PCR System (Applied Biosystems) and Power SYBR Green PCR Master Mix (Applied Biosystems). Relative quantification of mRNA was calculated by 2^-ΔΔCt. Data were normalized to the geometric average of Actb, Gapdh and Ppia and reported as fold changes compared to values obtained from the control group (set at 1.0). The list of primers is available as Figure 3—source data 2.

RNA was isolated from ∼50 mg of mouse quadriceps tissue in 1 mL of TRIzol (Invitrogen) using a TissueLyser (Qiagen) according to the manufacturer’s instructions. The RNA was resuspended into 50 µL DEPC treated water (Bioline). RNA was purified and DNAseI (Qiagen) treated using RNeasy Mini protocol (Qiagen). Quality and quantity was assessed using a 2100 Bioanalyser (Agilent Technologies) 6000 RNA kit (Agilent Technologies) according to the manufacturer’s instructions. One microgram (1 µg) of RNA was reverse transcribed using Super-Script™ III Reverse Transcriptase (Invitrogen), random pdN6 primers (Roche), 0.1 mM DTT (Invitrogen) and 10 mM dNTP (Invitrogen) in a 20 µL volume for 90 minutes at 50°C on a Veriti 96-well thermocycler (Applied Biosystems). All cDNA was diluted 1∶5 in DEPC treated water (Bioline) prior to RT-qPCR. Except for the Rn18S and Gapdh, the cDNA were diluted to 1∶100 due to the high abundance of these transcripts. Diluted cDNA was aliquoted into working volumes of 10 µl and stored at -80 °C until use. A maximum of three freeze-thaw cycles were performed for each aliquot.

Total RNA was isolated with miRNeasy Mini Kit (Qiagen), cDNA was synthesized with random p(dN)6 primers (Roche) and MMLV reverse transcriptase (Invitrogen). Real‐time PCR analysis in triplicates was performed with SYBR Green FastMix (Quanta) reaction mix. Primers used for RT–PCR, see Appendix Table S1.

Human breast microstructures and mouse organoids were homogenized in Trizol (Invitrogen). Aqueous phase containing RNA was chloroform extracted and processed with miRNeasy extraction kit (Qiagen). cDNA was synthesized using random p(dN)6 primers (Roche) and MMLV reverse transcriptase (Invitrogen). SYBR Green PCR Core Reagent System (Qiagen) was used for semi‐quantitative real time (RT‐PCR) using the following primers.

Gene	Forward primer	Reverse primer
M 36B4	GTG TGT CTG CAG ATC GGG TA	CAG ATG GAT CAG CCA GGA AG
M RANKL	CCC ACA ATG TGT TGC AGT TC	TGT ACT TTC GAG CGC AGA TG
M WNT4	AGG AGT GCC AAT ACC AGT TCC	CAG TTC TCC ACT GCT GCA TG
HPRT	GAC CAG TCA ACA GGG GAC AT	CCT GAC CAA GGA AAG CAA AG
GAPDH	CCC CAC TTG ATT TTG GAG GGA	AGG GCT GCT TTT AAC TCT GGT
CSN3	AAC AAC CAG CAT GCC ATG AG	AAC AAC CAG CAT GCC ATG AG
CSN1S1	GAG GCT TCT CAT TCT CAC CTG TC	ACT GCT CTC TGA TGG ATT CTG AAG
LALBA	AGG TCC CTC AGT CAA GGA ACA	GGC TTT ATG GGC CAA CCA GT

Mammary glands were homogenized with TRIzol reagent (Invitrogen), total RNA was isolated with miRNeasy Mini Kit (Qiagen), cDNA was synthesized with random p(dN)₆ primers (Roche) and MMLV reverse transcriptase (Invitrogen). Real-time PCR analysis in triplicates was performed with SYBR Green FastMix (Quanta) reaction mix. Primers used for RT-PCR, see Supplementary Table 5.

Cell pellets, tumor-bearing mammary glands, and microdissected lung, liver, and brain micrometastases were homogenized with TRIzol reagent (Invitrogen), total RNA was isolated with miRNeasy Mini Kit (Qiagen), cDNA was synthesized with random p(dN)₆ primers (Roche) and MMLV reverse transcriptase (Invitrogen). RT-PCR analysis in triplicates was performed with SYBR Green FastMix (Quanta) reaction mix and analyzed on QuantStudio 6 and 7 Flex Real-Time PCR System Software. Supplementary Table 3 provides the list of primers.